Method of identifying heteromultimeric modified ubiquitin proteins possessing ability to bind with ligands

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology and can be used for the identification of heteromultimeric ubiquitins possessing an ability to bind with a ligand-antigen. The method includes the contact of a totality of heterodimeric modified ubiquitins, including two ubiquitin monomers, bound to each other by a head-to-tail scheme, with the potential ligand in a display way. Each of the said monomers is modified in a different way and contains 5-8 substitutions in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 SEQ ID NO:1. After that, a heterodimeric modified protein, which has bound with the ligand with the binding affinity Kd in the range of 10-7-10-12 M and the monovalent binding activity. Claimed are DNA-libraries, responsible for obtaining a population of the said heteromultimeric ubiquitins, as well as libraries of proteins, obtained by the expression of the said DNA-libraries.

EFFECT: invention makes it possible to obtain the novel bonding proteins based on heteromultimeric ubiquitin, capable of specific high affinity binding with the selected ligands.

8 cl, 17 dwg, 4 ex

 

AREA of TECHNOLOGY

The present invention relates to a method of identification heteropolymers of ubiquitin with the ability to contact the ligand. In addition, the invention provides DNA libraries responsible for getting collectively referred to hetero-multimeric ubiquitin proteins and libraries of proteins obtained by expression mentioned DNA libraries, cells and phages containing the aforementioned DNA or proteins, polynucleotides responsible for getting referred a composite of proteins, and vectors comprising the aforementioned polynucleotides. In addition the proposed new binding proteins on the basis of heteropolymeric of ubiquitin able to specifically contact with high affinity-selected ligands.

Background of the INVENTION

There is a growing demand for a binding molecule composed of amino acids that are not immunoglobulins. Although the present antibodies are the best studied class of binding molecules, there is still a need for new binding molecules to obtain ligands with high affinity and specificity, since the molecules of immunoglobulins have major shortcomings. Although they can easily get and they can be directed to almost any target, they have very complicated molecular structure. Things�there is a continuing need to replace antibodies in smaller molecules which were easy to work with. These alternative linking agents can be profitably used, for example, in medical diagnosis, prevention and treatment of diseases.

Proteins that have a relatively specific three-dimensional structure, usually called protein cages can be used as a starting material for constructing these alternative binding agents. These frames usually contain one or more areas that are affected by specific or random change in the sequence, and such a randomization sequence is often performed to obtain a library of proteins, which can be selected for specific binding molecule. Molecules smaller than antibodies and with comparable or even more than a good affinity to the target antigen will be, as expected, better antibodies mean pharmacokinetic properties and immunogenicity.

In several previous approaches do use protein cages as source material for binding proteins. For example, in document WO 99/16873 describes the developed modified proteins of the family of lipocalin (so-called anticalin) exhibiting binding activity of some ligands. The structure of peptides of the family of lipocalin is modified by substituted�th amino acids in their natural binding pocket, using the methods of genetic engineering. Like antibodies, these anticline can be used to identify binding or molecular structures. Similar to antibodies, modify the structure of the flexible loop; these modifications give the ability to recognize ligands that are different from natural.

In document WO 01/04144 described the artificial creation of a binding domain on the surface of the protein in proteins with the structure of beta-Plast as those that lack the binding site.Through this newly created artificial binding domain can be obtained, for example, changes in γ-crystallin is a structural protein of the eye lens - which interact with ligands with high affinity and specificity. In contrast to the modification of binding sites, which are already present and formed from structures with a flexible loop that mentioned above for anticalins, these domains linking recreated on the surface of beta-layers. However, in document WO 01/04144 describes only the changes are relatively large proteins to create new binding properties. Because of their size, proteins according to WO 01/04144 can be modified at the level of genetic engineering only in ways that require some effort. In addition, in the proteins disclosed still, only a relatively small percentage to�I modified all the amino acids to maintain the overall structure of the protein. Therefore, only a relatively small surface region of the protein available for binding properties that did not exist previously. Moreover, WO 01/04144 reveals only the creation of properties of binding to γ-crystallin.

In document WO 04/106368 described the creation of artificial binding proteins on the basis of ubiquitin proteins. Ubiquitin is a small, Monomeric and cytosolic protein that is highly preserved in the sequence and is present in all known eukaryotic cells from protozoa to vertebrates organisms. In the body it plays a very important role in the regulation of the controlled degradation of cellular proteins. For this purpose proteins destined for degradation by co-valent contact ubiquitinate or polyubiquitinated chains during their passage through the cascade of enzymes to selectively degrade because of this label. According to the latest results, ubiquitin or tagging of proteins with ubiquitin, respectively, also plays an important role in other cellular processes, such as the import of several proteins or gene regulation.

In addition to the elucidation of its physiological function, ubiquitin is the subject of research mainly because of its structural and protein-chemical properties. The polypeptide chain of ubiquitin 7b consists of amino acids arranged in h�zvychaino compact α/β structure (Vijay-Kumar (Vijay-Kumar), 1987): almost 87% of the polypeptide chain is involved in the formation of secondary structural elements by means of hydrogen bonds. Secondary structures are alpha 3,5-spiral coils, as well as antiparallel beta-layer, consisting of four strands. The characteristic arrangement of these elements is antiparallel beta-Plast open protein surface on the rear side which Packed the alpha helix, which lies vertically on its top is generally considered the so-called ubiquitin-like motif styling. Another structural feature is the pronounced hydrophobic region of the protein between an alpha helix and a beta reservoir.

Because of its small size artificial preparation of ubiquitin can be carried out both by chemical synthesis or by biotechnological methods. Because of the favorable properties of stacking ubiquitin can be obtained by genetic engineering using microorganisms such as Escherichia coli, in relatively large quantities or in the cytosol or in the periplasmic space. Due to the oxidizing conditions prevailing in the periplasm, the latter strategy is usually reserved for the receipt of secretory proteins. For a simple and effective bacterial preparation of ubiquitin can be used as a partner in the unite for others, we get in�family of proteins, the receipt of which causes difficulties. By Association with the ubiquitin is possible to achieve high solubility and, through this, increased product yield.

Compared with antibodies or other alternative frameworks, artificial binding proteins based on ubiquitin proteins (also called Affilin®) have many advantages: small size, high stability, high affinity, high specificity, cost-effective microbial production and regulation of serum half-life. However, there is still a need for further development of these proteins in terms of new therapeutic approaches with a high degree of affinity to specific targets. Although in document WO 05/05730 described in General terms the use of frameworks of ubiquitin to produce artificial binding proteins, there is not a proposed solution, how to modify ubiquitinate protein to get specific and high-affinity binding with ligands like haptens and antigens, e.g., proteins and peptides and their epitopes. The methods described in WO 05/05730, monomers are modified by the ubiquitin protein or related proteins modified ubiquitin. Related forms create by screening and selection of one, two or more modified ubiquitin proteins with last�blowing their Association genetic or chemical methods, to obtain related forms that allow, for example, multispecific binding of ligands of different types of molecule one linked ubiquitin. In one example described site-directed binding of two identical proteins on the basis of ubiquitin (homodimers) to increase binding affinity compared to one molecule of the modified ubiquitin.

The purpose of the present invention is to provide a method of identifying multimeric ubiquitin proteins with a high ability to bind to the ligand. Another objective of the present invention is to provide a method of identification of new binding proteins based on modified ubiquitin that is able to specifically contact with high affinity-selected ligands.

The above objective is solved by the subject matter of the attached independent claims of the claims. Preferred embodiments of the invention are included in the dependent claims and in the following description, examples and figures in the drawings.

Disclosure of the INVENTION

More specifically, the inventors propose a method of identification heteropolymeric modified ubiquitin with the ability to bind to a ligand, comprising the following steps:

(a) providing aggregate Goethe�multimeric modified ubiquitin proteins originating from Monomeric modified ubiquitin proteins, and mentioned the set includes heteropolymeric proteins containing two or more different modified monomer of ubiquitin or at least one modified monomer of ubiquitin linked scheme head to tail, and at least two of each of the above-mentioned monomer mentioned heteropolymeric protein is differently modified by substitutions of surface amino acids in the open at least three amino acids located in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO:1, over and above, a modified Monomeric protein has amino acid sequence identity of at least 80% or at least 90% or at least 95% with unmodified ubiquitinated protein;

(b) providing a potential ligand for the aggregate of differently modified proteins;

c) the contact mentioned together differently modified proteins with said ligand;

(d) identification of the modified heteropolymer of protein through the screening process, and mentioned modified multimeric protein is bound with said ligand with a specific binding affinity of Kd in the range of 10-7-10-12M and manifested odnomomentno� binding activity against mentioned whether dictionary; and the selection

(e) isolation mentioned heteropolymeric modified ubique-vitanovac protein with said binding affinity.

Definitions of important terms used in this application

The term "ubiquitinate protein comprises ubiquitin in accordance with SEQ ID NO:1 and its modifications according to the following definition. Ubiquitin highly retained in eukaryotic organisms. For example, all mammals studied to date, ubiquitin has an identical amino acid sequence. Especially preferred are molecules of ubiquitin from human, rodents, pigs and primates. In addition, you can use ubiquitin from any other eukarioticheskogo source. For example, ubiquitin yeast differs only in three amino acids from the sequence SEQ ID NO:1. In General, ubiquitinate proteins covered referred to by the term "ubiquitinate protein" indicate amino acid identity of more than 70%, preferably more than 75% or more than 80%, more than 85%, more than 90%, more than 95%, more than 96% or until sequence identity of 97% with SEQ ID NO:1.

To determine the degree of sequence identity is derived of ubiquitin to the amino acid sequence of SEQ ID NO:1 can be used, for example, the program SIM local similarities� (Sawkin Huang and Webb Miller (Xiaoquin Huang and Webb Miller), "Developments in applied mathematics. Volume 12: 337-357, 1991) or Clustal W. (Clustal, W.) (Thompson and others (Thompson et al.). Nucl Acids Res., 22(22): 4673-4680, 1994.). Preferably, the degree of identity to the sequence of the modified protein with SEQ ID NO:1 is determined relative to the full sequence of SEQ ID N0:1.

In the present description of the invention, the terms "ligand" and "target" and "binding partner" are used interchangeably and can replace each other. A ligand is any molecule that is able to communicate with the affinity, which is defined here, with heteropolymers modified ubiquitinated protein.

"Heteropolymeric composite protein" or "heteropolymeric protein" of the present invention is considered to be a protein which includes one or more differently modified Monomeric ubiquitin proteins. "Heteropolymer" of the present invention, therefore, is considered to be a merger of at least two differently modified Monomeric ubiquitin proteins with two interacting domain areas, together creating a property monovalent binding to the specific binding partner. Preferred heterodimer or heterotrimer.

According to the invention, two differently modified monomer of ubiquitin that bind to one ligand must be connected by connecting "�tin-to-tail" with each other using, for example, genetic methods. These different modified combined monomers of ubiquitin bind monovalent and effective only if both "region binding domain" ("GSO") are acting together. These modified and associated ubiquitinate monomers, which form heteromeric protein bind to the same epitope by one continuous region of binding. This continuous region hetero-measure is formed by both determining the binding areas of at least two modules formed by at least two differently modified ubiquitinate monomers.

"The connection head-to-tail" must be understood as the merging of the two proteins by connecting them in the direction of the N-C-N-C-, depending on the number of links contained in multimer. When this compound head-to-tail monomers of ubiquitin can be directly linked without any linker. Alternatively, coupling of ubiquitin monomers can be performed by means of linkers, for example, the linker having at least the amino acid sequence GIG or having at least the amino acid sequence of SGGGG or any other linker, for example GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG or SGGGGSGGGG. Known in the art and can be used and other linkers to the genetic connection of two mono�'erov of ubiquitin. In General, heteropolymers unmodified ubiquitinate proteins produced by fusion of two, three or more differently modified Monomeric ubiquitin proteins, to obtain a compound of the modified protein of ubiquitin monomers. In yet another embodiment of the at least one of the monomers of ubiquitin is not modified, whereas at least one of other molecules of ubiquitin modified.

The term "population" refers to the library, which is a mixture of heterogeneous polypeptides encoded heterogeneous nucleic acids. The library consists of elements that have a single polypeptide encoded nucleinate sequence. Differences in the sequences between the elements of the library are responsible for the diversity present in the library. The library may take the form of a simple mixture of polypeptides or nucleic acids or in the form of organisms or cells, such as bacteria, viruses, animal or plant cells, etc., transformed with a library of nucleic acids. Preferably, each individual organism or cell contains only one element of the library. Preferably, the nucleic acid introduced into expression vectors to allow the production of polypeptides encoded by nucleic acids. Therefore�in one preferred aspect of the library can take the form of a population of organisms-owners, moreover, each organism containing one or more copies of the expression vector containing one element of the library in the form of nucleic acid, which can be expressed to produce its corresponding polypeptide member. Thus, a population of organisms-owners has the potential to encode a large range of genetically different variants of the polypeptides.

Such a population (set) heteropolymeric ubiquitin protein get, for example, by genetic fusion DNA libraries, each of which is responsible for differently modified Monomeric proteins, or in the alternative, in which the modified at least one of the Monomeric ubiquitin proteins, for the translation of DNA in heteropolymers integral proteins, displaying such proteins are displayed and checking the presence of proteins modified by heteropolymeric ubiquitin proteins containing monomer ubiquitinate proteins, which are interconnected according to the scheme head to tail, over and above modified heteropolymers ubiquitinate proteins bind with said ligand with a specific binding affinity to KD in the range of 10-7-10-12M and exhibit monovalent binding activity with respect to the above ligand. In order to obtain hetero�dimeric ubiquitinate protein connect the two DNA libraries that meet or get everyone, or at least one differently modified Monomeric protein to get heterotrimeric ubiquitinate protein, connect the three DNA libraries responsible for receiving each or at least one differently modified Monomeric protein, etc. To provide libraries for screening can be used and other alternatives. One example is the chemical synthesis of proteins, for example, solid state technology, and the introduction of variations in their amino acid composition. The specialist may consider and find useful, and other options. Therefore, the invention should not be construed as limited to the examples described in this document.

The invention accordingly discloses a method by which you get a range of polypeptides according to the functionality that is defined by the ability to bind the ligand, and a sub-group of polypeptides obtained as a result of choice, then apply for further cycles of selection according to ability to bind the target ligand to accumulate and increase the binding capacity of the ligand.

The invention allows the person skilled in the art to remove from the selected range of those polypeptides that are unable to communicate with the target ligand with CP�the rotary, defined in the claims. The invention allows the person skilled in the art to expand the selected range of those polypeptides that are functional and meet the requirements for the affinity.

One of the most important aspects of the present invention lies in the choice of modified heteropolymeric ubiquitin proteins with monovalent binding affinity to the target and subsequent determination of the modified amino acids responsible for binding affinity.

Another advantage of multimerization, preferably the dimerization, is to increase the number of amino acid residues that can be modified to create a new high-affinity binding properties. The advantage is that although modify even more amino acids, protein-chemical integrity is maintained without reducing the overall stability of the frame referred to the newly created binding protein for the purpose. On the one hand, the cumulative number of residues that can be modified to create a new binding site for the target increases, because the modified residues may be distributed across two, or three, or more Monomeric ubiquitinated proteins. The number of modifications thus may be two or x times larger than the number of modifier�the studied molecules of Monomeric ubiquitin. As a result, the modular structure of the binding protein on the basis of ubiquitin allows to increase the total number of modified amino acids, as mentioned modified amino acids are included in two, or three, or more molecules of Monomeric ubiquitin. The present method provides for the identification of molecules heteropolymers of ubiquitin having one monovalent specificity for a single epitope).

"Monovalent" should be understood as the possibility that the two connecting areas created in the first and second (and, optionally, subsequent) Monomeric unit the modified dimeric (optional make-up or, in General, multimeric) of ubiquitin, together bind ED-B synergistically and are United, i.e., both connecting areas work together to form a monovalent binding activity. If you take each separately connecting the first region and the second modified ubiquitin mentioned in heterodimeric the molecule, this region is obviously going to bind ED-B with much less efficiency and affinity than the dimeric molecule. Both connecting areas form a unique binding site that is formed as a continuous region of amino acids on the surface. heterodimeric modified ubiquitinated protein so mentioned modificarea�tion ubiquitin able to communicate much more effectively with ED-B than each Monomeric protein, taken separately. It is particularly important that, according to the present invention two Monomeric protein not linked after screening the strongest binding molecules of ubiquitin, but the screening process is performed in the presence heterodimeric of ubiquitin. After obtaining sequence information on the most strongly binding of ubiquitin molecules, these molecules can be obtained in any other way, for example, by chemical synthesis or methods of genetic engineering, for example, by linking already identified two of the ubiquitin monomer units together. It is understood that all examples presented here for dimeric modified ubiquitin proteins can also be modified for make-up or, in General, a multimeric modified ubiquitinated protein.

Thus, the use of heteropolymers, in particular heterodimers that share a common binding site for binding partners, opens the possibility of introducing an increased number of modified residues that do not impact negatively on protein-chemical integrity of the final binding molecules, since the total number of these modified residues distributed across two or more Monomeric units, which form a di - or multimer. Mentioned heteropolymers modified�s ubiquitinate proteins are present in the library of proteins.

After, for example, at least two different DNA libraries responsible for the production of modified Monomeric ubiquitin proteins were created by different modifications of selected codons in each of the Monomeric ubiquitin units, these libraries are genetically combine to produce DNA molecules responsible for getting heteropolymeric modified ubiquitin proteins. The DNA of these libraries is broadcast in proteins, and obtained by this modified heteropolymeric proteins brought into contact according to the invention with a target molecule to provide the binding partners to each other if a binding affinity exists.Preferred modified ubiquitin is heterodimer.

A very important aspect of the present invention is that the process of contact and screening perform, in relation heteropolymer, for example, heterodimers modified ubiquitinated protein. This process allows screening for those ubiquitin proteins, which provide a monovalent binding activity with the target.

It is particularly important that, according to the present invention, the monomer modified ubiquitinate proteins do not associate with each other after selection by screening the strongest binding molecules ubiquiti�and, but that is already a screening process performed in the presence heteropolymeric of ubiquitin. However, it should be noted that after receiving the information about the sequence for the strongest binding molecules heteropolymers of these ubiquitin molecules can be obtained in any other way, for example, by chemical synthesis or methods of genetic engineering, for example, by linking two already identified Monomeric ubiquitin links together to form heterodimeric binding protein.

According to the invention the contact is preferably performed using a suitable method of representation and choice, such as phage display, ribosomal display, TAT phage display, RNA display or display surfaces of cells that display surfaces of yeast or display surfaces of bacteria, preferably by phage display method. For completeness of disclosure reference is also made to the following publications: Hoess, Curr. Opin. Struct. Biol. 3 (1993), 572-579; Wells and Lowmann, Curr. Opin. Struct. Biol. 2 (1992), 597-604; Kay et al., Phage Display ofPeptides and Proteins - A Laboratory Manual (1996), Academic Press. The above methods known to experts in this field and can be used according to the invention, including their modifications.

Determining whether the modified protein is calculated quantitatively the binding affinity for ha�of the pre-defined binding partner can be accomplished, according to the invention, preferably one or more of the following methods: ELISA, spectroscopy a method of plasma surface resonance, fluorescence spectroscopy, the excited fluorescence of the sorted cells (FACS), isothermal titration calorimetry and analytical ultracentrifugation. Specialist General knowledge can be used and other methods available in this field.

In this document under "heteropolymer" refers to a protein that includes at least two different Monomeric ubiquitin protein. "Heterodimers" of the present invention is the fusion of two differently modified Monomeric ubiquitin proteins. They both show the United monovalent binding property for specific binding partner. It should be emphasized that multimeric modified, for example, dimeric, a ligand-binding ubiquitinate protein of the present invention is not obtained by screening of each separate monomer ubiquitinated protein and combining at least two of them later, and by screening for identifying multimeric, at the option of dimeric proteins consisting of the first and second or further Monomeric level, which together exhibit monovalent binding activity with the mentioned leagues�Ndom. It should be expected that each of these subzones, shows quite a limited binding affinity to the ligand, whereas only the United multimeric or dimeric modified ubiquitinate protein will have an excellent binding properties described herein.

In one embodiment, the implementation of the method relates to the identification of the modified heterodimeric ubiquitinated protein, in which two Monomeric ubiquitin link connected according to the scheme head to tail, with each monomer mentioned dimeric protein is differently modified by substitution of at least 3, preferably 6, amino acids in positions 2,4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO:1 (each of them superficially open)

moreover, the above-mentioned substitution include:

(1) in the first Monomeric unit substitution, at least in amino acid positions 6, 8, 63, 64, 65 and 66;and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally in amino acid position 2; or

(2) in the first Monomeric unit substitution, at least in amino acid positions 2, 4, 6, 62, 63, 64, 65 and 66; and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally in amino acid position 2; and, by the choice�, other modification, preferably a substitution of other amino acids, over and above the level of the modified Monomeric ubiquitin has amino acid identity with SEQ ID NO:1 at least one of the group of 80%, at least 83%, at least 85%, at least 83% and at least 90%, and mentioned protein has specific binding affinity with a ligand Kd=10-7-10-12M and exhibits a monovalent binding activity with respect to the above ligand.

In other embodiments of the present invention, 6, 7, 8, 9 or all of the amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO:1 is modified in each Monomeric ubiquitin unit. It is understood that the present invention permits the consolidation of each of these variations in each of the monomer units, for example, in the first and second link. For example, the first monomer unit may include 6 modifications, whereas the second link includes 7 or 8 modifications, the first link may include 8 modifications and the second link 7 modifications etc. Each of these amino acids may be selected in the first and the second link, and both links then join. Preferred substitution is described below.

DETAILED description of the PRESENT INVENTION, the Methods of display modified heteropolymeric ubiquitin protein

Procedures phage�first and ribosomal display, adapted to the present application is described below and in the Examples section. They cited as examples of the selection procedure according to the invention for the detection of variations of ubiquitin, which show the properties of binding of the potential ligand described herein. In the same way can be applied, for example, how to represent the bacteria (bacterial surface display; Daugherty et al., 1998, Protein Eng. 11(9):825-832) or yeast cells (yeast surface display; Kieke et al., 1997 Protein Eng. 10(11): 1303-10) or cell-free system of choice, such as ribosomal display (Hanes and Pluckthun, 1997, Proc Natl Acad Sci USA. 94(10):4937-4942; He and Taussig, 1997_Nucleic 5 Acids Res. 25(24):5132-5134) or cis display (Odegrip et al, 2004 Proc Natl Acad Sci USA. 101(9):2806-2810) or mRNA display. In the latter case, a temporary physical linkage of genotype and phenotype is achieved by connecting the variations of the protein with a suitable mRNA through the ribosome.

In the procedure described here phage display recombinant variations of ubiquitin are presented on the filamentous phage, whereas encoding DNA presents variations are presented at the same time Packed in single-stranded form in ragovoy shell. Thus, in the framework of affine enrichment variations that have certain properties, can be selected from the library, and their genetic information can be amplified by infection of suitable bacteria or EXT�go to another cycle of enrichment, respectively. Representation of the mutated ubiquitin on the surface of phage is achieved through a genetic Association with a signal sequence from the amino end, preferably with Re In the signal sequence, and the capsid or preferred surface protein of the phage is carboxyterminal Association with capsid protein FMC or its fragment. In addition, the obtained composite protein may contain other functional elements, such as a label affinity or epitope antibodies for the detection and/or purification affinity chromatography, or the recognition sequence of a protease-specific cleavage of a compound of protein in the affinity enrichment. In addition, the UAG terminator can be represented, for example, between the genome for variations of ubiquitin and the coding region of the capsid protein of the phage or its fragment, which is not recognized during translation in a suitable suppressor strain, partly due to the introduction of a single amino acid.

Bacterial vector, suitable for the selection procedure in the context of the isolation of the variations of ubiquitin with the properties of binding to the target and asked which introduced gene cassette described for compound protein, called Femidom. Among others, it contains the intergenic region of the filamentous phage (e.g. M13 or fl) or part of it, to�area in the case of a superinfection of the bacterial cells, carrier phagemid by phages-helper cells, such as, for example, MC, leads to the packaging of the Cova turbulent closed DNA strands phagemid in the phage capsid. Created thus phage particles are secreted by bacteria and represent the corresponding variation of ubiquitin coded because of its Association with capsid protein pIII or its fragment on their surface. Native capsid protein pIII are present in ragovoy particle, so that its ability to reinfiltrate suitable bacterial strains and, therefore, the ability to amplificatoare corresponding DNA remains. Thus, the physical link between the phenotype variations of ubiquitin, i.e., its potential binding property, and its genotype.

The obtained phage particles can be selected relative to the binding of the variations of ubiquitin with any target, e.g., ED-B, TNF-alpha, MIA-2, NGF, IgG and through other ways known to specialists in this field. For this purpose presents variations of ubiquitin can be temporarily immobilized on the target substance, and associated, for example, in microtiter tablets, and can be specifically-eluted after separation is not communicating variations. The elution is preferably performed by alkaline solutions, such as, for example, 100 mm triethylamine. Alternative, al�probing can be performed in acidic conditions, by proteolysis or direct upload infected with bacteria. Phage particles, thus obtained, can be reamplification and enriched by successive cycles of selection and amplification variations of ubiquitin with binding properties, e.g., ED-B, TNF-alpha, MIA-2, NGF, IgG or any other targets.

One option phage display is a method Tat-phage display (Paschke, M. and V. Honea (Paschke, M. and W. Hohne (2005)). Gene 350(1): 79-88; see also EP 1567643). In this method variant of ubiquitin, which is encoded by Femidom, the governed through a system of double-arginine translocation (Tat) that exports folded proteins, which has already reached its native conformation already. in the cytoplasm (Bruser (Bruser) 2007 Appl Microbiol Biotechnol 76(1): 35-45). Requirement for secretion is merging with specific N-terminal signal pepti-house, which sends this variant of ubiquitin from the time Tat. After logging in Periplus-MATIC space N-terminal signal peptide is removed by a signal peptidase. In the periplasmic space variant of ubiquitin is then covalently connects with the capsid protein pIII or its C-terminal fragment, which gets secreted from the cytoplasm towards the Sec, as well as other proteins of the phage. This connection between ubiquitin and pIII is implemented by high-affinity interaction W�of ppera Leikin Jun N-end of the protein pIII and the zipper Leikin Fos at the C-end variant of ubiquitin. Additional cysteines at the N and endings With each of the zippers Leikin allow covalent bond between the two proteins and, as a consequence, they also allow for covalent bond between display ubiquitin and the product of its encoding gene in ragovoy particle.

Further characterization of the variations of ubiquitin, thus obtained, can be made in the form phagemid, i.e., combined with phage, or after cloning of the corresponding gene cassette expressing in a suitable vector in the form of soluble protein. Suitable methods known in the art or described in the literature. Characterization may include, for example, determining the DNA sequence and, thus, the primary sequence of isolated variations. In addition, the affinity and specificity of isolated variations can be used to detect standard biochemical methods such as ELISA or spectroscopy surface plasmon resonance, displacement chromatography, fluorescence anisotropy, FACS, isothermal titration calorimetry, analytical ultracentrifugation. In light of the stability analysis, for example, spectroscopic methods in connection with chemical or physical deployment known to specialists in this field. Other good� known methods are CD spectroscopy, protein fluorescence spectroscopy and NMR spectroscopy.

In yet another embodiment, variations of ubiquitin procedures for ribosomal display is prepared by a cell-free system of transcription/translation, and present as a complex with the corresponding mRNA and the ribosome. For this purpose, a DNA library, which is described above, is used as the basis in which the genes of variations are present in the form of Association with appropriate regulatory sequences for expression and protein biosynthesis. Due to the deletion of the termination codon at the end of the 3' gene libraries, as well as suitable experimental conditions (low temperature, high concentration of Mg2+) three-part complex consisting of emerging protein, mRNA and ribosomes, is maintained during transcription/translation in vitro.

After creating a library of proteins containing heterodimers modified ubiquitinate proteins by different modifications of selected amino acids in each monomer unit of ubiquitin-modified dimeric proteins brought into contact according to the invention, with a target to allow partners to communicate with each other if there exists a binding affinity. These libraries of proteins can be in the form of a display library display method, or can be used�you any other way representing modified proteins in such a way as to ensure contact between the modified protein and the protein target, and mentioned display method is, by choice, rahovym display, ribosomal display, rahovym display TAT, yeast display, bacterial display or mRNA display.

Potential ligands and targets modified by heteropolymeric ubiquitin protein

The present invention was successfully tested on the following representative antigens: ED-B, TNF-alpha, MIA-2, NGF and IgG. It is understood that these antigens were chosen only to show that the methods described here can be successfully implemented by a person skilled in the art without undue difficulties after receipt of the information presented here. The invention is not limited to these specific antigens, and can be executed on all or at least most of the ligands and molecules-targets known in this field. These targets can be selected by a specialist within his General knowledge in this area. Below is a General definition of ligands and targets, as well as antigens and haptens, and provides some examples of other potential target molecules.

According to the invention, the term "antigen" refers to a substance capable of being bound described in the estuaries�ü modified ubiquitin, the function of which is comparable with the antibody. Used here an alternative term is "ligands", "binding partner" or "target". Modified ubiquitinate proteins of the present invention provide binding molecules that act like an antibody, in the same time without having its drawbacks. The term "antigen" includes haptens, peptides, proteins, sugars, DNA, etc. From the publication Roche Lexikon Medizin, 4th edition; Urban &Fischer/Elsevier GmbH) can be taken, the following definitions of antigen and hapten, which are also used in this description.

Antigen (AG): Identification of any substance recognized as foreign ("own") the immune system. Initiates in most cases the immune reaction that leads to the immune system (="immunogen"), in the case of Allergy (="allergen"), and atopy ("utopian"), respectively, this immune response is too high. AG induces humoral (reaction antigen-antibody) and/or cellular defense response (see "immunity" below). If AG-tolerated by the immune system (immune tolerance), it is also called "tolerogen". Effective as an antigen in the main complex and high-molecular substances (of body protein, polysaccharides, nucleotides, and many synthetic compounds) having a chemically identifiable features (determinants) that are responsible for immune response. Classifierwidget 1) a complete AG, for the most part with a high molecular weight and is able to trigger an immune response, 2) as hapten low molecular weight (=semi-antigen), which acts as an immunogen only after he connected with a larger molecule carrier. Is called, for example, Xeno-, ALLO - or isogenic, autologous AG; auto-, hetero-, transplantation, anti-tumor viral AG.

Hapten: simple low molecular weight chemical compound responsible for the specificity of antigen (AG) or is able to specifically communicate with the antibody because of its structure (determinants), respectively, but is unable to create allergies: as opposed to the full AG. He becomes a complete antigen after binding to belkom body, called "medium".

"Ligand" or "target" or "binding partner" is a molecule that is recognized by the described modified getremotename the ubique-votinovym proteins. Examples of ligands that can be used in the practice of the present invention include, but are not limited to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral EPI-tops, hormones, hormone receptors, polypeptides, peptides, enzymes, substrates for enzymes, cofactors, drugs (e.g., opiates, steroids, etc.), aspect�; sugars, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

To sum up, as a binding partner for modified proteins, proposed according to the invention, can be used all dietary and medical active and relevant molecules. Possible binding partners will be described below as an example. It should be said, however, that this list may be added some other possible ligands. Like the relationship between antibody and antigen, a list of potential binding partners may be supplemented with other potential ligands.

In the present invention examples of binding partners for heterodimeric of ubiquitin are extradosed b of fibronectin (ED-B), cytokine (tumor necrosis factor alpha (TNF-α), MIA-2, immunoglobulin, or portion thereof, e.g., a whole antibody (for example, immunoglobulin), and growth factor (e.g., NGF, for example, nerve growth factor). Below are brief descriptions of these ligands. However, it should be emphasized that all these ligands are well known in this area for many years and known to experts in the relevant fields of technology. Therefore, the following descriptions will only briefly outline some of the important parameters of these proteins, the amino acid posledovatelno�and which is also known.

Extradosed B (ED-B) of fibronectin is a small domain, which is administered by alternative splicing of the primary transcript RNA molecule fibronectin. It is known that ED-B is involved mainly in cancer and psoriasis. Surprisingly, high levels of expression of ED-B were detected in the areas of primary lesions, and metastasis of many solid cancer sites in humans, including breast, non-small cell lung, colorectal, pancreatic, skin, hepatocellular, increased intracranial meningioma, glioblastoma (Menrad and Menssen (Menrad u. Menssen), 2005). In addition, ED-B may be associated with diagnostic agents and can advantageously be used as diagnostic tools. One example is its use in molecular imaging, for example, atherosclerotic plaques and the detection of cancer, for example, by immunoscintigraphy cancer patients. Also many other possible ways of use in diagnosis.

Amino acid sequence of 91 amino acids of human extradata. In (ED-B) of fibronectin is shown in SEQ ID NO:2. For protein expression have to add the starting methionine. ED-B is abundantly present in mammals, e.g., rodents, cattle, primates, carnivores, etc. Examples of animals in which there is a 100% ID�tecnost sequence with ED-B man are Rattus norvegicus, Bos taurus, Mus musculus, Equus caballus, Macaca mulatto, Canis lupus familiaris, and Pan troglodytes.

Protein MIA ("the activity, inhibiting melanoma, also called CD-RAP Junior. čaemyj of cartilage is sensitive to retinoic acid protein") protein is expressed in chondrocytes and was originally isolated because of its antiproliferative properties in vitro. It was originally detected in the supernatant of cell culture of melanoma cells and isolated therefrom. After purification and partial sequencing of this protein, the cDNA fragment MIA person was isolated using degenerate primers and RT-PCR (polymerase chain reaction reverse transcriptase). Now known sequences MIA for human, mouse, ox, rat and striped Pertini. Related protein, MIA-2, described in documents EP 1410803 B1 and US-2010/0212037. These documents are incorporated herein by reference.

The tumor necrosis factor alpha (TNF-alpha), a pleiotropic cytokine, is produced mainly by macrophages, but it and produce other types of cells. TNF-alpha demonstrates useful and pathological activity. It has as effects that stimulate the growth and properties that inhibit the growth, besides being self-regulating. Useful functions of TNF-alpha include the maintenance of homeostasis by regulating the circadian rhythm of the body, creating an immune response to bacterial, viral, �Rybnoye and parasitic infection replacement or reconstruction of damaged tissue by stimulating fibroblast growth and, as the name already implies, the elimination of some tumors. TNF-alpha is involved as a mediator in a large number of diseases.

Nerve growth factor (NGF) is secretively protein, which was opened over 50 ago as a molecule that promotes the survival and differentiation of sensitive and sympathetic neurons. NGF is a member of a family of neuro-trophic factors known as the neurotrophins. NGF binds with high affinity to a kinase receptor tropomyosin, known as TrkA. NGF is also able to bind the receptor, known as P75, a member of the superfamily of receptors of tumor necrosis factor, which also interacts with other neurotrophins. Beta-a chain of NGF is responsible for the activity of NGF, stimulating the growth of nerves. This beta-chain homodimerization and enters into a larger protein complex. The structure and function of the NGF considered, for example, in the following publications: Sofroniew M. V. and others (Sofroniew, M. V. et al.) (2001) Annu. Rev. Newosci. 24:1217-1281; Weisman and DeVos (Weismann, S. and de Vos), A. M. (2001) Cell. Mol. Life Sci. 58:748-759; Fahnestock M. (Fahnestock, M.) (1991) Curr. Top.Microbiol. Immunol. 165:1-26.

IgG antibodies are large molecules of about 150 kDa composed of 4 peptide chains. They contain 2 identical heavy chains of about 50 kDa and 2 identical light chains approaching�Uo to 25 kDa, having, thus, tetrameric Quaternary structure. Two heavy chains are linked together with a light chain disulfide bonds. The resulting tetramer has two identical halves, which together form a Y-shape. Each end of this fork contains an identical binding site of the antigen. Region in the IgG Fc have a highly conservative site of N-glycosylation. N-glycans attached to this SAI that are mainly corporationbank centennaire structures of the complex type. In addition, small amounts of these N-glycans are also area bisection GlcNAc and α-2,6 linked sialic acid residues.

Methods of selection, enrichment and characterization of proteins display

The choice heteropolymeric modified by ubiquitin in relation to their activity of binding to a specific ligand with a specific binding affinity of Kd in the range of 10-7-10-12M can be made by methods known to experts in this field. For this purpose presents variations of ubiquitin can be temporarily immobilized on the target substance, and associated, for example, in microtiter tablets, or may be associated with magnetic particles after binding in solution, respectively. After separation nesvezhije genetic variation information is variation with regard�yuusha activity can be specifically-eluted in the form of mRNA through degradation of the ribosomal complex. The elution is preferably performed using EDTA. The thus obtained mRNA can be isolated and reverse transcribed into DNA using suitable methods (reverse transcriptase reaction), and the thus obtained DNA can be reamplification.

Through successive cycles of transcription/translation, selection and amplification variations can enrich variations of ubiquitin with binding properties with a predetermined hapten or antigen.

Further characterization of the variations of ubiquitin, thus obtained, can be made in the form of soluble protein, as detailed above, after cloning of the corresponding gene cassette expressing in a suitable vector. Suitable methods known in the art or described in the literature.

Preferably, the step for detecting proteins having binding affinity to specific binding partner, the next step of isolation and/or enrichment of another protein.

After expression ubiquitinated protein, modified according to the invention, it may be further purified and enriched by methods known as such. Choose the methods depend on several factors, known as such to the experts in this field, for example, used expressing vector addresses�and host the target area using the amount of protein and other factors. To simplify purification of the protein, modified according to the invention may be combined with other peptide sequences with a high affinity with the materials division. Preferably selected such associations, which have no adverse effect on the functionality ubiquitinated protein or can be separated after cleaning due to the introduction of specific cleavage sites of the protease. Such methods are also known as such to specialists in this field.

Unmodified and modified ubiquitinate proteins as a starting point for mutagenesis

The terms "protein, able to bind" or "binding protein" refers to ubiquitination protein with one binding region, which is described below. The binding region may include at least two regions, determining binding ("EP"), Each monomer has at least one area defining binding; at least two of the monomer form of multimer having at least two regions, determining the binding, which form one region binding to a single antigen. Any such binding protein on the basis of ubiquitin may include additional protein domains that are not binding domains such as, for example, fluid multimerization, polypeptide tags, polypeptide linkers and/or non-protein molecules are polymers. Some examples of non-protein molecules polymers are gidroksiètilovyh starch, polyethylene glycol, polypropylene glycol or polyoxyalkylene.

Further multimerization heteropolymeric modified ubiquitin proteins can also be accomplished by post-translational merge heteropolymeric modified ubiquitinated protein with effector molecules, having the domain multimerization (e.g. TNF-α). In yet another embodiment of the further multimerization is accomplished by the use of polybag.-singlecolor (PEG) linker. In yet another variant implementation of the domain multimerization also acts as a pharmaceutically active component; one example is TNF-alpha, which acts as the domain multimerization, and as a pharmaceutical component.

Modified heteropolymers ubiquitinate proteins

The term "modified ubiquitinate protein" refers to modifications to ubiquitinated protein of any one of substitution, insertions or deletions of amino acids or combinations thereof, wherein the substitution are the most preferred modifications which may be supplemented with any one of the modifications described above. Number�about modifications strictly limited, as mentioned modified Monomeric ubiquitinate links have amino acid identity with SEQ ID NO:1 at least one of the group of 80%, at least 83%, at least 85%, at least 83% and at least 90%. At most, the total number of substitutions in the Monomeric unit, therefore, is limited to 15 amino acids corresponding to 80% amino acid identity. Other alternatives are 13, 12,11, 10, 9, 8, 7, 6 or 5 modified amino acids. The total number of modified amino acids in heterodimeric molecule of ubiquitin is 30 amino acids, corresponding to 20% of amino acid modifications on the basis heterodimeric protein. Other alternatives are 28, 26, 24, 22, 20, 18, 16, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 modified amino acids in the dimeric molecule of ubiquitin. Amino acid identity dimeric modified ubiquitinated protein compared to the dimeric ubiquitin consisting of two unmodified Monomeric ubiquitin proteins with a basic monomer sequence SEQ ID NO:1 is selected at least one from the group of 80%, at least 83%, at least 85%, at least 83% and at least 90%.

Modified ubiquitinate proteins, obtained by the method of the present invention, constructed with new communicating sredstvami to the molecule target illigant or binding molecule (these expressions are used here interchangeably).

The term "substitution" also includes chemical modifications of amino acids by, for example, substitution or addition of chemical groups or residues to an original amino acid. The amino acid substitution in at least one region with an open surface proteins, including amino acids located in at least one strand of the beta-layer region of the beta reservoir or facility to 3 amino acids near the thread of the beta reservoir is very important.

The modification performed by methods that are well established and well known in this field. "Non-specific modified nucleotide or amino acid sequence" is a nucleotide or amino acid sequence, which in several positions was subjected to an insertion, deletion or substitution of nucleotides or amino acids, the nature of which cannot be predicted. In many cases, the introduced non-specific nucleotides (amino acids), or a sequence of nucleotides (amino acids) will be "completely non-specific" (e.g., as a result of a randomized synthesis or PCR-mediated mutagenesis). However, non-specific sequences may also include sequences that have a common functional feature (e.g., the ability to react with the ligand of the expression product), or nonspecific seq�coherence can be nonspecific in the sense what is the end product of the expression is completely non-specific sequence, for example, with a uniform distribution of the different amino acids.

In order to enter the randomized fragments into the vectors correctly, according to the invention should preferably be non-specific nucleotides were introduced into the expression vector according to the principle of site-directed, PCR-mediated mutagenesis. However, the known specialist and other options, and, for example, you can enter a library of synthetic non-specific sequences in the vectors.

To create mutants or libraries by combining PCR can be performed, for example, three PCR reactions. Two PCR reactions performed to generate partially overlapping the intermediate fragments. A third PCR reaction is performed to merge the intermediate fragments.

Method of constructing a library or mutant variants may include constructing a first set of primers around the desired restriction site (primer restriction site), direct and reverse primer restriction and a second set of primers around, for example, in front and behind the codon of interest (mutagenic primers), forward and reverse mutagenic primer. In one embodiment of the primers design immediately in front of and behind, respectively, the codon�m, of interest. Restriction and mutagenic primers used for construction of the first intermediate and second intermediate fragments. Two PCR reactions create these intermediate linear fragments. Each of these intermediate linear fragments includes at least one mutated codon of interest flanking the nucleotide sequence and the cleavage site. A third PCR reaction using these two intermediate fragments and the forward and reverse primers restriction enzymes for the production of the joint linear product. On the contrary, still the loose ends of the linear product was digested with restriction enzymes to create sticky ends on the linear product. The sticky ends of the linear product combine by using DNA ligase to obtain a ring product, for example, a circular polynucleotide sequence.

To construct the intermediate fragments perform the design and synthesis of two sets of forward and reverse primers, wherein the first set contains the cleavage site of restriction enzyme together with its flanking nucleotide sequence, and the second set contains at least one variant of the codon. of interest (mutagenic primers). Specialists in this field will understand that the number�STV variants will depend on the number of options is desirable modifications of amino acids. The author of the present invention provides that if in this process will be used other restriction enzymes, the exact location of the cleavage site and a corresponding sequence of forward and reverse primers may be modified accordingly. The level of technology available other ways, and they can be used is outlined.

In addition to the introduction of randomized fragment of the expression product in the frame in accordance with the present invention, it is often necessary to combine non-specific sequence with a partner on the merger by combining a randomised nucleotide sequence with a nucleotide sequence that encodes at least one partner in the Association. Such partner Association may, for example, to facilitate expression and/or purification/isolation, and/or further stabilization of product expression.

For the purpose of cleaning a partner in the Association may include cleaning, such as a His6 tag, label Thue, the target sequence for biotinidase BSP, BirA, label flu, lacZ, and GST. In addition, partner in the Association may include a sorting signal or sequence against the target.

The amino acid substitution to create a new binding domain specific for a target molecule, can be performed� according to the invention with any desired amino acid, i.e., to modify, to create a new binding property to the molecule-targets, it is not necessary to take care that the amino acids had any particular chemical property or side chain, respectively, which are similar to that of the replaced amino acids, so for this purpose you can use any desired amino acid.

The phase modification of the selected amino acids is performed, according to the invention, preferably by mutation at the genetic level by non-specific mutagenesis, i.e., non-specific substitution of selected amino acids. Preferably, the modification of ubiquitin perform the methods of genetic engineering to modify the DNA belonging to the corresponding protein. Preferably, the expression of the ubique-vitanovac then carry out protein in prokaryotic or eukaryotic organisms.

Substitution will, in particular, the amino acids on the open surface of the four beta strands of the beta reservoir or amino acids with an open surface to 3 amino acids near the thread of ubiquitin protein strands of the beta reservoir ubiquitinated protein. Every beta thread usually consists of 5-7 amino acids. With reference to SEQ ID NO:1, for example, beta-strands usually include amino acid residues 2-7, 12-16, 41-45 and 65-71. Areas that can be optionally and preferably modified to include p�position up to 3 amino acids (i.e., 1, 2 or 3) next to the thread of the beta reservoir. The preferred areas, which can be optionally and preferably modified to include, in particular, amino acid residues 8-11, 62-64 and 72-75. Preferred fields include beta-turns, linking two beta-strands together. One preferred beta stage includes amino acid residues 62-64. The most preferred amino acid that is located near the thread of the beta reservoir, is the amino acid in position 8. Furthermore, other preferred examples of amino-acid substitutions are 36, 44, 70 and/or 71. For example, those areas which can be optionally and preferably modified to include amino acids 62, 63 and 64 (3 amino acids), or 72, 73 (2 amino acids), or 8 (1 amino acid).

The number of amino acids that can be added or withdrawn, limited 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids in the monomer subzone of ubiquitin and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26 or 28 amino acids relative to dimeric ubiquitination squirrel in x times the number of modifications in the Monomeric protein. Typically, the number of inserts in the Monomeric molecule comprises 1-10 amino acids and/or 1-7 deletions of amino acids. The number of substitutions is at least 6 and at most 14 substitutions of amino acids in the monomer molecule. Dimeric molecule in�includes in General at least 12 and at most 28 substitutions and/or in General at least one and at most 20 insertions, and/or at least one and at most 14 deletions. You can use all integers within these ranges, and they are covered by the invention, and all combinations of the number of deletions, insertions and substitutions are possible provided that the overall structural integrity of the molecule is preserved. In one embodiment of the present invention preserves the structure of the beta-layer.

In other embodiments, amino acid residues are modified by substitution of amino acids. However, it is also permissible deletions and insertions. The number of amino acids that can be added or withdrawn, limited 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in Monomeric subzone of ubiquitin and respectively 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 amino acids relative to dimeric ubiquitination squirrel. In one embodiment of the insertions of amino acids do not. In yet another embodiment, the implementation does not perform deletions.

Provided that the modified ubiquitinate protein of the present invention includes in addition to the aforementioned substitutions defined in the claims and explained here, as well as deletions and/or additions of one or more amino acid position of amino acids for the human ubiquitin neatening type (SEQ ID NO:1) must be aligned with modifiziert�NYM by ubiquitin, to highlight proteins corresponding to each other. In the case of composite proteins (see below), the numbering (and alignment) of each of subzones monomer of ubiquitin is carried out identically, i.e., alignment of, for example, dimer begin in position 1 of the amino acid for each respective subzone.

In Monomeric ubiquitinated protein, preferably a mammalian, e.g. human, at least 10% of the amino acids present in beta strands or positions up to 3 amino acids near the thread of the beta-layer, preferably at least 20%, more preferably at least 25%, can be modified, preferably substituted. As a maximum, preferably approximately 50% of the amino acids present in beta strands or positions up to 3 amino acids near the thread of the beta-layer, more preferably at least about 40%, or about 35%, or to about 30%, or to approximately 25% are modified, preferably substituted. In one of the beta threads usually modified 1-4 amino acids. In one embodiment, the implementation of two of the six amino acids preferably in the first and fourth β strands, for example, in the field of amino acid residues 2-7 or 65-71, modified.

Modified Monomeric ubiquitin according to the invention, used as a building W�eno for heteropolymer, totals in the aggregate to 20% of the amino acids that need to be modified. Given this, there exists a sequence identity with SEQ ID NO:1 modified ubiquitinated protein at least 80%. In other embodiments, the sequence identity on amino acid level is at least 83%, at least 85%, at least 87% and, in addition, at least 90%, at least 92% or at least 95% sequence identity with an amino acid sequence of SEQ ID NO:1. The invention also encompasses the identity of the amino acid sequence of the modified ubiquitinated protein more than 97% compared to the amino acid sequence of SEQ ID NO:1.

In yet another embodiment of the present invention have already pre-modified ubiquitin (in which modified 3, or 4, or 5, or 6, or 7 amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and/or 68 of SEQ ID NO:1) is used as a starting point for further modifications, to create the property of binding with the target, and can be obtained ubiquitin, which in the aggregate to 9, 10, 11, 12, 13, 14 and a maximum of 15 amino acids of ubiquitin SEQ ID NO:1 are modified, preferably substituted. For example, further modifications may include modifications in the amino acids 74 and 75 or the amino acid�OTE 45, to create increased stability or improved protein-chemical properties. According to one example, a modified Monomeric ubiquitin as a construction element for heteropolymeric protein can be obtained in this way with 14 substitutions and deletion of the aggregate number of amino acids of ubiquitin, which corresponds to approximately 20%. It was completely unexpected, since it is usually much smaller percentage is enough to disrupt the packing of the protein.

In one embodiment, these amino acids are modified to create a field with new binding properties, which form a continuous region on the surface of the protein. Thus can be created in a continuous area that has a property of binding with ligand-targeted. "Continuous region" according to the invention applies to the following: because of the charge, the spatial structure and hydrophobicity/hydrophilicity of its side chains of amino acids interact with their environment accordingly. This environment can be a solvent, usually water, or other molecules, for example, spatially close amino acids. By structural information about the protein, as well as the corresponding software can be characterized surface proteins. For example, the area on�arnosti section between the protein atoms and solvent can be visualized this way, including information about how structured this area of the interface, which parts of the surface accessible to solvent, or as distributed on the surface charges. Continuous region may be disclosed, for example, the visualization of this type using appropriate software. Such methods are known to those skilled in the art. According to the invention, basically, the entire area with an open surface can be used as a continuous area on the surface that you want to modify to create new binding properties. In one embodiment, the implementation for this purpose, the modification may also include alpha-helical region. In heterodimers modified ubiquitinated protein region that defines a binding that includes two of the areas with an open surface forming together a single continuous region, which includes two lengths of one area, which determines the binding.

Modification of amino acids in at least one region of a protein with an outdoor surface, comprising at least one beta-thread region of the beta reservoir or positions up to 3 amino acids near the thread of the beta layer is very important. "Structure of beta-Plast" is defined as essentially plastopodobnye and almost fully stretched. In contrast to alpha-SP�the Raleigh, formed of undisturbed segment of the polypeptide chain, beta-layers can be formed in different regions of the polypeptide chain. This image region, which are separated farther from each other in the primary structure, can enter into close proximity with each other. Beta thread usually has a length of 5-10 amino acids (usually 5-6 residues in ubiquitin) and has almost fully stretched conformation. Beta-strands are suitable so close to each other that are formed by hydrogen bonds between the-0 one thread and the NH group of the other strand and Vice versa. Beta-layers can be formed from several threads and have plastopodobnye structure in which the position of the alpha-C atoms alternates between positions above and below the reservoir plane. Side chains of amino acids follow this model and, thus, an alternative point up or down. Depending on the orientation of the beta-strands, the layers are classified into parallel and antiparallel. According to the invention, both types can mutate and be used for preparation of the proposed proteins.

For mutagenesis of beta-strands and beta structure-formation in the ubiquitin choose the beta thread. or positions up to 3 amino acids adjacent to the beta thread (which is thread beta formation), which are close to the surface. Amino acids with an open surface can be identified in relation � available x-ray crystallographic structure. If the crystal structure is not available, you can make attempts to predict through computer analysis of the region of the beta layer on the open surface and the availability of individual provisions of amino acids with regard to the available primary structure or model the three-dimensional structure of the protein and to obtain information about potential amino acids with an open surface. Further disclosure of this question can take, for example, in J. IO 1. BioL, 1987 Apr 5; 194(3):531-44. Vijay-Kumar S, C. E. Bugg, W. J. Cook

However, you can also make modifications in the beta-layer or positions up to 3 amino acids adjacent to the beta thread, for which time-consuming pre-selection provisions of amino acids for mutagenesis can be omitted. Region of DNA that encode patterns of beta-layers, or up to 3 amino acids near the thread of the beta reservoir isolated from the DNA of the environment, is subjected to non-specific Muta-Genesis and then re-integrate into the DNA coding for the protein from which they were removed previously. This is followed by the process of selection of mutants with desired binding properties.

In yet another embodiment the beta-strands or up to 3 amino acids adjacent to the beta thread close to the surface is chosen as above and identify the position of amino acids for mutagenesis in these selected areas. Position�of amino acids, chosen in such a way can then be subjected to mutagenesis at the DNA level or by directed to the site of mutation, i.e., a codon encoding a specific amino acid is replaced by a codon encoding another previously selected specific amino acid, or the substitution is carried out in the context of non-specific mutagenesis, which defines the substitutable position of the amino acid, but not codon, encoding a new, yet to a certain amino acid.

"Amino acids from the open surface are amino acids that are accessible to the surrounding solvent. If the availability of amino acids in the protein of more than 8% compared to the availability of amino acids in the model Tripeptide Gly-X-Gly, the amino acids referred to as "exposed surface". These areas of the protein, or position of individual amino acids, respectively, are also preferred binding sites for potential binding partners, which selection must be made according to the invention. In addition, reference is made to the publication of Custer and others (Caster et al, Science 1983, 221, 709-713, and Sraka and Ripley (Shrake &Rupley), 1973 J. Mol. Biol. 79(2):351-371, which for full disclosure included in this application by reference.

Variations of ubiquitin characterized by substitutions of amino acids in artificial binding site, newly created from bear�raising protein and from each other, can be created by targeted mutagenesis of the respective sequence segments. In this case, amino acids having properties such as polarity, charge, solubility, hydrophobicity or hydrophilicity can be replaced or substituted, respectively, the amino acids with the corresponding other properties. In addition to the substitutions, the terms "mutation" and "modified" and "substituted" also include insertions and deletions. On the protein level modifications can also be made by chemical alteration of the side chains of amino acids according to methods known to experts in this field.

Methods mutagenesis of ubiquitin

As a starting point for mutagenesis of the corresponding sequence segments can be used, for example, cDNA of ubiquitin, which may be prepared, modified and amplified by methods known to experts in this field. Commercially available reagents and methods for site-specific changes of ubiquitin in relatively small regions of the primary sequence (approximately 1-3 amino acids) ("Quick Change", the company Stratagene; set "Mutagene Phagemid in vitro Mutagenesis Kit", the company Biorad). For site-directed mutagenesis larger areas experts can specific variants of implementation, for example, poly�arisna chain reaction (PCR). For this purpose you can use a mixture of synthetic oligodeoxynucleotides having compositions degenerate base pairs in the desired positions, for example, to enter mutations. This can also be done through the use of analogues of base pairs, which in natural form are not found in genomic DNA, such as, for example, inosine.

The starting point for mutagenesis of one or more beta strands of the beta field-layer or positions up to 3 amino acids near the thread of the beta layer may be, for example, cDNA of ubiquitin or genomic DNA. In addition, the gene coding for ubiquitin-new protein can also be prepared synthetically.

In one embodiment, mutagenesis is performed by assembling oligonucleotides of DNA that carry the NNK codon amino acids. You should understand, however, that you can use other codons (triplets). Mutations performed in such a way as to preferably maintain the structure of the beta-layer. Typically, the mutagenesis occurs on the outside of the stable region of the beta reservoir, open on the surface of the protein. This includes both site-specific and non-specific mutagenesis. Site-specific mutagenesis, including a relatively small region in the primary structure of the (approximately 3-5 amino acids) may be created using commercially available Noboa�s Stratagene® (QuickChange®) or Bio-Rad® (phagemid) Mutagene® kit for mutagenesis in vitro) (see US 5,789,166; US 4,873,192). If site-specific mutagenesis is subjected over a wider area, shall be prepared DNA cassette in which the area for the mutagenesis produced by assembling nucleotides containing mutant and unmodified provisions (Nord, etc. (Nord et al), 1997 Nat. Biotechnol. 8, 772-777; McConnell and Hess (McConell and Hoess), 1995 J. Mol. Biol. 250, 460-470.). Non-specific mutagenesis can be introduced through the distribution of DNA in strains of gene-mutator or amplification PCR (error-prone PCR) (for example, Pannekoek, etc. (Pannekoek et al.), 1993 Gene 128, 135 to 140). For this purpose, a polymerase with increased frequency of errors. To enhance the degree of entered mutagenesis or to combine different mutations, respectively, mutations in PCR fragments can be combined by DNA rearrangements (Stemmer (Stemmer), 1994, Nature 370, 389-391). An overview of these mutagenesis strategies in relation to the enzymes included in this review Kuchner and Arnold (Kuchner and Arnold) (1997) TIBTECH 15, 523-530. To implement this non-specific mutagenesis in the selected region of DNA must also be designed DNA cassette, which is used for mutagenesis.

Various procedures known as such and available for mutagenesis are ways for site-specific mutagenesis, methods for non-specific mutagenesis, mutagenesis with the use�against PCR or similar methods.

In one preferred embodiment of the present invention pre-determine the position of amino acids for mutagenesis. The choice of amino acids for modification is carried out in accordance with the restrictions of paragraph 1 of the claims against those amino acids that must be modified. In each case usually create a different library of mutants, which is subjected to screening using methods known as such. Usually a pre-selection of amino acids for modification can be particularly easy to perform when sufficient structural information has ubiquitinated protein to be modified.

Methods for targeted mutagenesis, and mutagenesis of longer sequence segments, for example by PCR, chemical mutagenesis or by using bacterial strains gene mutator also known in the art and can be used according to the invention.

In one embodiment, mutagenesis is performed by assembling oligonucleotides of DNA that carry the codon NNK. amino acids. You should understand, however, that you can use other codons (triplets). Mutations performed in such a way as to preferably maintain the structure of the beta-layer. Typically, the mutagenesis occurs on the outside of the stable region of beta-place�and, open on the surface of the protein. This includes both site-specific and non-specific mutagenesis. Site-specific mutagenesis, including a relatively small region in the primary structure of the (approximately 3-5 amino acids) may be created using commercially available kits Stratagene® (QuickChange®) or Bio-Rad® (phagemid Mutagene® kit for mutagenesis in vitro) (see US 5,789,166; US 4,873,192).

If site-specific mutagenesis is subjected over a wider area, shall be prepared DNA cassette in which the area for the mutagenesis produced by assembling nucleotides containing mutant and unmodified provisions (Nord, etc. (Nord et al), 1997 Nat. Biotechnol. 8, 772-777; McConnell and Hess (McConell and Hoess), 1995 J. Mol. Biol. 250, 460-470.). Non-specific mutagenesis can be introduced through the distribution of DNA in strains of gene-mutator or amplification PCR (error-prone PCR) (for example, Pannekoek, etc. (Pannekoek et al.), 1993 Gene 128, 135 to 140).

For this purpose, a polymerase with increased frequency of errors. To enhance the degree of entered mutagenesis or to combine different mutations, respectively, mutations in PCR fragments can be combined by DNA rearrangements (Stemmer (Stemmer), 1994, Nature 370, 389-391). An overview of these mutagenesis strategies in relation to the enzymes included in this review Kuchner and Arnold (Kuchner and Arnold) (1997) TIBTECH 15, 523-530. To implement this, respec�cific mutagenesis in the selected region of DNA must also be designed DNA cassette, used for mutagenesis.

Nonspecific substitution according to one example of the present invention is at least 6 amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and/or 68 of the monomer of ubiquitin can be performed particularly easily by means of PCR, since these provisions are localized close to the amino or carboxy end of the protein. Accordingly, the codons for manipulating located at the 5' and 3' end of the corresponding cDNA strands. Thus, the first oligodeoxynucleotide used for the mutagenic PCR reactions except codons at positions 2, 4, 6 and/or 8, which will mutate, corresponds to the sequence of the coding strands for the amino-end of ubiquitin. Accordingly, the second oligodeoxynucleotide - except codons at positions 62, 63, 64, 65, 66 and/or 68, which will mutate, at least in part corresponds to non-coding strands of the polypeptide sequence of the carboxy-end. Through both oligodeoxynucleotides polymerase chain reaction may be performed using a DNA sequence that encodes a Monomeric ubiquitin, as a template.

In addition, the obtained amplification product can be added to a different polymerase chain reaction using flanking oligodeoxynucleotides, which is administered, for example, consistently�ti recognition for restriction endonucleases. According to the invention is preferably obtained gene cassette in a vector system suitable for use in a subsequent selection procedure for isolation of variations of ubiquitin with binding properties to a predetermined hapten or antigen.

The amino acid substitution to create a new binding domain specific for the selected ligand can be made according to the invention with any desired amino acid, i.e., to modify, to create a new property of binding with a selected ligand, it is not necessary to take care that the amino acids had any particular chemical property or side chain, respectively, which are similar to that of the replaced amino acids, so for this purpose you can use any desired amino acid.

The phase modification of the selected amino acids is performed, according to the invention, preferably by mutation at the genetic level by non-specific mutagenesis, i.e., non-specific substitution of selected amino acids. Preferably, the modification of ubiquitin perform the methods of genetic engineering to modify the DNA belonging to the corresponding protein. Preferably, the expression ubiquitinated then carry out protein in prokaryotic or eukaryotic organisms.

With�publicly to the invention, modified ubiquitinate protein can also be preferably prepared by chemical synthesis. In preferred embodiments, amino acid residues are modified by substitution of amino acids.However, it is also permissible deletions and insertions. By choice, the number of amino acid insertions or deletions is 1-10, 1-5, 2, 3 or 4 amino acids. In one embodiment, the implementation of insertion of amino acid is not carried out. In yet another embodiment, the implementation does not perform deletions.

After performing the above modifications, the inventors have found amino acid sequence of the modified ubiquitin, as described in the examples which bind their targets with very high affinity (Kd values up to 10-10M).

The areas to be modified ubiquitin

Areas for modification can be primarily selected based on the fact that they may be available to selected binding partner, and from the fact that the overall structure of the protein is expected to show tolerance to the modification.

In addition to modifications in surface open beta strands, can also be carried out modifications in other open surface areas of the protein, preferably in positions up to 3 amino acids adjacent to the beta thread. These modified region participate in the newly covered by�Ohm binding with high affinity with the target.

According to another preferred embodiment of the present invention, at least 3 or 4 or 6, optionally at least 8, 10, 12 and maximum 15 open surface amino acids of ubiquitin, preferably of ubiquitin to the mammal or human, can be modified in a Monomeric ubiquitin, in which the modification is preferably a substitution. This includes the modification 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 open surface amino acids of ubiquitin. These at least 3 and a maximum of 15 open surface modified amino acids then form a region with binding affinity for a pre-specific binding partner. This area is defined here as the region binding domain" ("GSO"). In this respect, particularly preferably at least 2, optionally at least 4, then by choice of at least 6, 8, 10, 12 and maximum 15 open surface amino acids were located in the region of the beta-layer, i.e., in the thread of the beta reservoir, or were distributed among several beta-strands or up to 3 amino acids near the thread of the beta reservoir. Also preferably, at least 3 of all modified, preferably substituted, amino acids were located directly next to each other in the primary sequence.

In another one�m embodiment of the present invention the amino acid at one or two, preferably two, of the four beta-strands in the protein or in positions up to 3 amino acids, preferably near two of the four beta-strands is modified to create a new binding property. Also, optionally, can be carried out modification in three or four of the four beta strands or positions up to 3 amino acids adjacent to three or four beta-strands to create a binding to a selected target or ligand.

Particularly preferably, the amino acids in the threads of the amino end and the carboxy-end or positions up to 3 amino acids near the filament amino-end and the carboxy-end have been modified, preferably substituted to create a new binding site with the ligand or target. In this respect, particularly preferably up to 3 amino acids near the thread carboxymethyl-the end of the beta reservoir were modified, preferably substituted, and 1 amino acid near the filament amino-end of the beta reservoir was modified, preferably substituted.

According to the invention ubiquitin modified in its amino acids, preferably by substitution, into at least three amino acids in the following provisions of ubiquitin mammal, preferably human ubiquitin: 2, 4, 6, 8, 62, 63, 64, 65, 66, 68. These at least three amino acids from the groups of amino acids forming�Ute continuous surface open area on the surface of ubiquitin, found, are particularly suitable for the creation of modified proteins having binding affinity, which had not previously existed in relation to the specific binding partner, e.g., ED-B, TNF-alpha, NGF, IgG, MIA-2, or any other target. At least three of these amino acid residues should be modified. By choice, 3, 4, 5, 6, 7, 8, 9 or 10 of the mentioned amino acid residues are modified, preferably substituted, optionally, in combination with additional amino acid residues.

To determine the degree of sequence identity is derived of ubiquitin to the amino acid sequence of SEQ ID NO:1 can be used, for example, the program SIM local similarities (Sawkin Huang and Webb Miller (Xiaoquin Huang and Webb Miller), "Developments in applied mathematics. Volume 12: 337-357, 1991) or Clustal W. (Clustal, W.) (Thompson and others (Thompson et al.). Nucl Acids Res., 22(22): 4673-4680, 1994), which is available for free from the authors and their Institute for the analysis of the multiple alignment. Preferably, the degree of identity of the sequence derived from SEQ ID NO:1 is determined relative to the full sequence of SEQ ID NO:1.

Methods of determining the values of the binding affinity are known as such and can be selected, for example, of the following: ELISA, technology based on surface plasma resonant�(SDP) (proposed, for example, Biacore®), exclusion chromatography, fluorescence spectroscopy and isothermal titration calorimetry (ITC).

In another aspect the invention relates to a composite protein, including Goethe amultimedia binding protein of the present invention combined with a pharmaceutically and/or diagnostically active component; reference is made, for example, the document US 7,838,629, the entire contents of which is incorporated by reference.

Integral protein of the present invention may comprise a polypeptide components, for example, peptide linkers, peptide ligands, for example, a therapeutically or diagnostically relevant radionucleotides. It may also include small organic or amino acid compounds, for example, sugar, oligo - or polysaccharide, fatty acid, etc. In one preferred embodiment the binding molecule on the basis of heteromera of ubiquitin linked peptide having therapeutically or diagnostically relevant properties of the linker on the basis of the amino acid, or a ligand, or protein with a therapeutically or diagnostically relevant properties.

The specificity of binding (dissociation constants)

The specificity of binding protein component according to the invention are the same as defined above for the composition�CSOs protein expressed in kDa. In accordance with the invention, the term "kilodaltons" defines the specific binding affinity, which in accordance with the invention is in the range from 10-7up to 10-12M. a Value of 10-5M and below can be considered Express quantitatively the binding affinity. Depending on the application, a value of 10-7M - 10-11M is preferably, for example, for chromatographic applications or 10-9-10-12M for example, for diagnostic or therapeutic applications. Other preferred values of the binding affinities are in the range from 10-7up to 10-10M, preferably up to 10-11M.

Multimerization of ubiquitin

According to the invention, two differently modified monomer of ubiquitin, genetically related by combining "head to tail" bind to the same epitope of a target molecule, e.g., ED-B, TNF-alpha, IgG, Mia-2, NGF, or any other molecule-target, and is effective only if both the field binding domain act together. In other words, they bind to the same epitope through continuous binding region, which is formed by the joint action of both binding regions of the two monomers.

The monomers can be connected directly or via linkers. Appropriate the preferred�bubbled the linkers are linkers SEQ ID NO:32 or having at least the sequence GIG, or having at least sequence SGGGG, or any other linker, for example GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG or SGGGGSGGGG. However, there are many known linkers that can be used instead of these.

Library

In another aspect of the present invention is directed to a library containing the DNA responsible for getting Modific yovanny Monomeric ubiquitin proteins as defined above, which forms the basis for providing heteropolymeric, preferably heterodimeric, the ubiquitin proteins of the present invention.

In another aspect of the invention provides a library of associations containing DNA obtained by combining the two libraries, as mentioned above, each library is responsible for obtaining links differently modified Monomeric ubiquitin proteins to get heterodimerize integral proteins of ubiquitin, and their monomer units are linked at the location of the "head to tail". The library is responsible for obtaining heterodimeric protein component of ubiquitin exhibiting monovalent binding activity against a given target. This binding between a run or using any of the linkers known to the person skilled in the art, or a linker described herein. "R�EIT modified" also includes an alternative one unmodified molecules, present in heterodimers protein.

Example 1 describes the preparation of a complex library. However, caution should be exercised in relation to the quality of the library. The quality of the library in frame technology is the first, depending on its complexity (number of separate variants), and functionality (structural and protein-chemical integrity of the resulting candidates). Both features, however, can have negative effects on each other: the increasing complexity of the library by increasing the number of modified positions on the frame can lead to deterioration of protein-chemical characteristics of options. This can cause reduced solubility, aggregation and/or low product yield. The reason for this is the high deviation from the original frames, having energetically favorable packing of the protein.

Therefore, when designing such a library of frameworks requires a suitable balance between the extreme positions of the introduction of the greatest possible number of variations in the original sequence to optimize it for the target and, on the other hand, to keep the original primary sequence to the maximum extent to avoid negative protein-chemical effects.

Specific modifications in heterodimeric ubiquiti�new proteins

Heterodimer of ubiquitin according to the invention that binds to a ligand with Kd=10-7-10-12M and exhibits a monovalent binding activity with respect to the above ligand, selected from the following two alternatives:

(1) in the first Monomeric unit substitution, at least in amino acid positions 6, 8, 63, 64, 65 and 66; and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally 2, and

(2) in the first Monomeric unit substitution, at least in amino acid positions 2, 4, 6, 62, 63, 64, 65 and 66; and

in the second Monomeric unit substitution, at least in amino acid positions 6, 8, 62, 63, 64, 65 and 66; optionally 2.

In one embodiment of the compound, the protein is genetically combined heterodimers mentioned monomer of ubiquitin, having a substitution in the amino acids in positions 6, 8,63-66 of the first monomer of ubiquitin and substitution in amino acid residues in positions 6, 8, 62-66, and optionally in position 2 of the second monomer of ubiquitin, preferably

- in the first monomer of ubiquitin replacement

lysine (K) to tryptophan (W) or phenylalanine (F) in regulation 6,

Leikin (L) to tryptophan or phenylalanine (W, F) in position 8,

lysine (K) to arginine (R) or histidine (H) at position 63,

glutamic acid (E) lysine (K),arginine (R) or histidine (H) at position 64,

series (S) to phenylalanine (F) or tryptophan (W) at position 65 and

threonine (T) to Proline (P) at position 66;

- in the second monomer of ubiquitin preferred substitution of lysine (K) to threonine (T), asparagine (N), serine (S) or glutamine (Q) at position 6,

Leikin (L) to glutamine (Q) or threonine (T) or asparagine (N) or serine (S) in position 8,

glutamine (Q) to tryptophan (W) or phenylalanine (F) at position 62,

lysine (K) to serine (S), threonine (T), asparagine (N) or glutamine (Q) at position 63,

glutamic acid (E) asparagine (N), serine (S), threonine (T), or glutamine (Q) in position 64,

serine (S) to phenylalanine (F) or tryptophan (W) at position 65, and

threonine (T) to glutamic acid (E) or aspartic acid (D) at position 66, and

by choice, glutamine (Q) to arginine (R), histidine (H) or lysine (K) in position 2.

These alternative substitution in each monomer may be combined with each other without any restrictions, provided that the resulting heterodimer modified ubiquitin have a specific activity of binding with the ligand KD=10-7-10-12M and are active monovalent binding to the ligand, and provided that the structural stability ubiquitinated protein is not destroyed or affected.

Most preferred are the following substitution

(1) in the first Monomeric unit at least K6W, L8W, K63R, EC, S65F and TR;

and in the second Monomeric unit at least K6T, L8Q, Q62W, K63S, E64N, S65W and TE; additionally Q2R, or

(2) in the first Monomeric unit at least Q2T, F4W, K6H, Q62N, K63F, EC, S65LHT66S;

and in the second Monomeric unit modification at least in the provisions 6, 8, 62, 63, 64, 65 and 66, in addition, optionally,

in the second Monomeric unit at least K6X, L8X, Q62X, K63X, EH, S65X and TH; additionally Q2X, where X can be any amino acid.

Particularly preferred are the following substitution in the first monomer of ubiquitin to generate proteins binding to ED-B:

2: Q□T, 4: F□W, 6: K□H, 62: Q□N, 63: K□F, 64: E□K, 65: S□L, 66: T□S.

To connect the two monomers "head to tail" can be use or not use the linker. Preferred are linkers SEQ ID NO:32 or a sequence GIG or SGGGGIG, or SGGGGSGGGGIG.

In one preferred embodiment of implementation, heterodimer of ubiquitin with two fields that determine the binding and acting together for ligand binding ED-B, includes the amino acid sequence of SEQ ID NO:33 or 34. Another preferred compound is a protein of the present invention, including TNF-alpha as a pharmaceutically active component, has the sequence of SEQ ID NO:35 or 36. In yet another embodiment, carried out�I heterodimer of ubiquitin with two fields determining the binding and acting jointly for ligand binding ED-B, includes the amino acid sequence of Fig. 11, corresponding to SEQ ID NO:XX.

Another preferred protein represented by the following sequence, where XXXX can be any amino acid (SEQ ID NO:47).

MTIWVHTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSD

YNINFKLSLHLVLRLRGGSGGGGSGGGGIG

MQIFVXTXTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSD

UNIXXXXXLHLVLRLRGG

Examples of proteins with these sequences is shown in Fig. 11. As the linker used here SGGGGSGGGGIG. It is understood that feasible alternatives is also another type of linker or no linker.

Polynucleotides, the host cell, the vectors of the present invention

In another aspect, the present invention also encompasses polynucleotides which are responsible for the creation of a protein or protein composite as described above. Additionally, the invention encompasses vectors comprising the aforementioned polynucleotide.

In an additional aspect of the present invention are covered by the host cell, including protein described herein or a compound of a protein and/or polynucleotide coding for the recombinant protein or protein composite of the present invention or the vector containing the polynucleotide.

The use of molecules modified heteromeric�dimensional ubiquitin

Modified ubiquitinate proteins of the present invention, capable of binding ligand with high affinity, should be used, for example, for the preparation of a diagnostic means for use in vitro or in vivo, and therapeutic agents. Proteins according to the invention can be used, for example, as molecules of a direct effector (modulator, antagonist, agonist) or antigen-recognizing domains.

In human and veterinary therapy and prevention pharmaceutically effective medications containing at least one heteromeric ubiquitinate protein, modified in accordance with the invention, for binding to ED-B can be prepared by ways of informing as such. Depending on herbal drug, these compositions can be administered by parenteral introduction in case of injection or infusion, systematically, rectally, intraperitoneally, intramuscularly, subcutaneously, intradermal or other traditionally used methods. Type pharmaceutical preparation depends on the type of disease, severity of disease, the patient and other factors known to specialists in the field of medicine.

Depending on the chosen partner for associations, pharmaceutical composition of the invention is adapted to focus on the treatment of cancer, e.g. breast and colorectal Rako�, or any other neoplastic disease with abundant expression of the target.

Compositions adapted for the maintenance of a therapeutically effective dose. The value assigned to the dose depends on the patient, type of disease, age and body weight of the patient, and other factors, known as such.

The compositions contain a pharmaceutically or diagnostically acceptable carrier and, optionally, can contain other auxiliary agents and fillers known per se. They include, for example, but without limitation, stabilizing agents, surfactants, salts, buffers, tinted agents, etc.

The pharmaceutical composition may be in the form of a liquid preparation, cream, lotion topicale applications, aerosols, powders, granules, tablets, suppositories or capsules, emulsions or liposomal preparation. The compositions are preferably sterile, non-pyrogenic and isotonic and contain traditional and pharmaceutically acceptable additives, known per se. Additionally, reference is made to the rules of the United States Pharmacopoeia or the publication "Pharmaceutical Sciences Remington", May Publishing Company (1990).

"Pharmaceutical composition" according to the invention can be present in the form of a composition in which different active ingredients and diluents and/or carriers are mixed with each others�d, or may take the form of a combined preparation, in which the active ingredients are present in partially or completely separate form. An example of such a composition or combined preparation is set.

"Composition" according to the present invention includes at least two pharmacologically active compounds. These compounds can be administered simultaneously or separately with a time interval from one minute to several days. Connections can be entered in the same way or in different ways; for example, oral administration of the same active compounds and parenteral introduction in case of introduction of a different connection. Also, the active compounds can be in the same medication, for example, in one solution for infusion or as a set containing both connections separately. It is also possible that both compounds were present in two or more packages.

In yet another embodiment of the pharmaceutical composition is in the form of a set of parts providing for a separate position for recombinant ubiquitinated protein/protein composite of the invention and one or more chemotherapeutic agents.

Modified ubiquitinate proteins according to the invention can be prepared by many traditional and well-known methods, such as simple strategy �organicheskoi synthesis, the solid-phase synthesis methods or commercially available automated synthesizers. On the other hand, they can also be prepared in a conventional recombinant methods as such or in combination with traditional methods of synthesis.

On the selection, modification can be accomplished by genetic engineering at the level of DNA and expression of the modified protein in prokaryotic or eukaryotic organisms or in vitro.

In yet another embodiment, the implementation stage modification includes the step of chemical synthesis.

In one aspect of the present invention mentioned set of differently modified proteins produced by the genetic Association of two DNA libraries, each of which is responsible for differently modified Monomeric ubiquitinate proteins.

BRIEF DESCRIPTION of the DRAWINGS

Fig. 1 shows, it is shown that the recombination front (first) modified monomer of ubiquitin with a scope that defines the binding, referred to as OOS, with differently modified rear (second) monomer of ubiquitin with a scope that defines the binding, referred to as OOS, for creating heterodimer leads to a significant increase in affinity and specificity of binding. The modified molecules of ubiquitin are analyzed through�Ohm Biacore, fluorescence anisotropy binding on cells and tissue sections. Shown concentration-dependent ELISA results (conc. - ELISA) binding of several options heterodimers of ubiquitin with ED-B person.

Fig. 1A shows the binding affinity Kd=9.4 µm =9,45×10-6M for monomer B (here: SPWF28-41B10-th). Shaded circles indicate the binding of the first monomer B with a fragment V that represents extradosed B(ED-B) of fibronectin. The control fragment 6789 contains ED-B and shown unfilled circles.

Fig. 1B shows binding affinity heterodimer of ubiquitin. Heterodimer contains first monomer V, combined with another, the second monomer that gives the option N (here: SPWF28-46H9-th). Binding affinity IN much increased compared with the monomer, shown in Fig. 1A due to monovalent binding of the two monomers with the target ED-B (Kd=131 nm =1,3×10-7M; here M, shaded circles). The control fragment 6789 contains ED-B and shown unfilled circles.

Fig. 2 shows the affinity and activity combined with cytokine molecules heterodimer binding ED-B, on the basis of the modified ubiquitin.

Fig. 2A shows a high affinity binding ED-B heterodimer N (Kd of 50.7 nm=5×10-8M) on the basis of the modified ubiquitin. Shaded circles show the concerned�tion N with EDB; as a negative control, the binding N with BSA (bovine serum albumin) (unfilled circles).

Fig. 2B shows high affinity binding ED-B heterodimer N on the basis of the modified ubiquitin, combined with the cytokine TNF-alpha for multimerization of heterodimer N (Kd=5,6 nm=5,6×10-9M).

Fig. 2C shows an analysis of examples of candidates selected from the library heterodimeric modified ubiquitin, for example, variants A, 22D1, N, V of heterodimer. The value of ELISA in kDa is increased to the target ED-B compared with the cytosolic fibronectin (c-FN), used as control, confirming the specific binding with the target.

Fig. 2D shows the results of the analysis of molecules A modified heterodimers of ubiquitin through analyses of interactions without labels using Biacore®. Analyzed different concentration options heterodimers of ubiquitin (see legend to the Figure: 0-15 µm E) for binding to ED-B immobilized on the chip (Biacore) to analyze the interaction between heterodimers option A and ED-B. the value in the kDa cannot be determined from the curve analysis of Association and dissociation.

Fig. 2E shows the results of the analysis of molecules 41 B10 modified heterodimeric ubiqui�in analyses of interactions without labels using Biacore®. Analyzed different concentration options heterodimers of ubiquitin (see legend to the Figure: 0-15 µm V) for binding to ED-B, immobilized on the chip (Biacore) to analyze the interaction between heterodimers option 41 B10 and ED-B. curve Analysis of Association and dissociation gave a KD value of 623 nm (6,2×10-7M).

Fig. 3 shows the contribution of different variants on the basis of the modified ubiquitin in binding affinity and specificity. These different versions share the modules of the overall sequence, which are marked with lowercase letters. The options were analyzed for their binding to ED-B. Fig. 3 shows other combinations of monomers giving heterodimer modified ubiquitin. Heterodimerize options 46-A5, 50-G11 and 46-N4 have the same first (front) modified monomer with OSD (marked "a" in this Figure), but the second (rear) monomer of ubiquitin modified OSD in different positions. Options 52-D10 and 52-B3 have another first (front) modified monomer, compared to 46-H9 with OOS, but the same second (rear) monomer of ubiquitin with OSD (marked "e").

Heterodimer modified ubiquitin have the following sequence:

46-H4: SEQ ID NO:25, 45-H9: SEQ ID NO:26, 46-A5: SEQ ID NO:27, 50-G11: SEQ ID NO:28, 52-B3: SEQ ID NO:29, 52-D10: SEQ ID NO:30

The above sequence�eljnosti have been modified during the experiments by adding tag His-Tag sequence LEHHHHHH (SEQ ID NO:31).

As can be seen from Fig. 3, 46-N4 has excellent binding affinity to ED-B (KD=189 nm); 46-A5 and 52-D10 does not have binding activity, whereas other modified ubiquitinate small proteins have binding activity with ED-B, compared to 46-H4. Thus we can conclude that both the monomer in heterodimers version required for high-affinity binding to the target: both monomers exhibit monovalent binding to the target.

Heterodimer modified ubiquitin with high activity of binding to ED-B, named N, identified the following substitutions of amino acids in both areas binding domain of two monomers in comparison with the monomers of ubiquitin wild type:

in the first module (OSD) (a) Q2G, F4V, K6R, Q62P, K63H, EA, S65T, T66L

in the second module (OSD) (e) K6H, L8M, Q62K, K63P, E64I, S65A, THE

50G11

in the first module (N)(a) Q2G, F4V, K6R, Q62P, K63H, ER, S65T, T66L

in the second module (C) K6M L8R, Q62M, K63N, EA, S65R, T66L

N

in the first module (N) (a) Q2G, F4V, K6R, Q62P, K63H, ER, S65T, T66L

in the second module (d) K6G. L8W, Q62T, K63Q, E64Q, S65T, T66R

V

in the first module (g) Q2R, F4P, K6Y, Q62P, K63P, E64F, S65A, T66R

in the second module (N) K6H, L8M, Q62K, K63P, E64I, S65A, THE

52D10 (not associated with ED-B)

in the first module Q2V, F4C, K6R, Q62T, K63A, ER, S65G, T66D

in the second module (N) (e) K6H, L8M, Q62K, K63P, E64I, S65A, THE

A (not associated with ED-B)

in the first module (N)(a) Q2G, F4V, K6R, Q62P, K63H,ER, S65T, T66L

in the second module (b) K6L, L8M, Q62L, K63A, E64F, S65A,

Fig. 4 shows the sequence alignment. Line 1: two monomer ubiquitinated wild-type protein (1st line) associated with a 12-amino acid linker SGGGGSGGGGIG starting at position 77 and ending at position 88; and a second monomer with OSD starts at position 89 with methionine. This dimeric ubiquitinates wild-type protein is aligned with heterodimers option 46-H9 modified ubiquitin (2nd line) with different modifications in the first and second monomers, giving two of the OSD. Both GSO act together in the binding of the target due to monovalent binding with the target.

Fig. 5 shows the sequence alignment heterodimeric variant 1041-D11 (1st line) modified with ubiquitin "Ub2_TsX9" (ubiquitin modified in position 45 in both monomers in tryptophan, showing the linker GIG between the two monomers (regulations 77 to 79; the second monomer starts with a methionine at position 80), and the substitution of glycine by alanine in the last C-terminal amino acids of the 2nd monomer. The third line shows "UBI-dt dimer, the dimer of ubiquitin wild type; not showing the alignment of the linker (thus, the second monomer starts at position 77 with methionine). 4 line shows "UBI-dt monomer", which is a human wild-type ubiquitin.

<> Fig. 6 shows the concentration-dependent ELISA of the binding of variant 1041-D11 heterodimers of ubiquitin with ED-B person. Variant 1041-D11 has a high-affinity binding to ED-B (KD=6,9 nm=6,9×10-9M). Closed circles show the binding affinity of variant 1041-D11 heterodimers of ubiquitin with ED-B containing a fragment of fibronectin (named 67B89-tO), compared to the lack of binding of this variant with negative control (named 6789-tO) (unfilled circles).

Fig. 7 shows the results of the competitive, concentration-dependent ELISA of the binding of variant 1041-D11 heterodimers of ubiquitin with immobilized ED-B containing a fragment of fibronectin (67 V89), in the presence of increasing amounts of free target. Variant 1041-D11 heterodimers of ubiquitin shows a very high-affinity binding to ED-B with IC50=140 nm soluble 67 V89, indicating that binding of 1041-D11 is not an artifact of the deterioration of the structural qualities of the ED-B due to immobilization on vibraphones surface used in the competitive ELISA settings.

Fig. 8 shows the result of the analysis molecule 1041-D11 modified heterodimers of ubiquitin in the analyses of interactions without labels using Biacore®. Analyzed different concentrations of this option heterodimers of ubiquitin (see legend in the Figure: 0-200 n� 1041-D11) for binding to ED-B, contains a fragment of fibronectin (named 67 V89) immobilized on the chip SA (Biacore). The curve analysis of Association and dissociation gave a KD value of 1 nm (1×10-9M) and the exponent koffOf 7.7×10-4with-1that shows a long half-life of the complex 1041-D11 and ED-B.

Fig. 9 shows the binding of variant 1041-D11 heterodimers of ubiquitin with the ED In in a concentration-dependent ELISA simultaneous analysis of serum stability of the binding activity. Shows different conditions, such as pre-incubation for 1 hour at 37°C this option in the serum of mice or rats in PBST as the control. The values in CD are in the range from 10 to 20 nm. Thus, we can conclude that the binding of heterodimer 1041-D11 with ED-In the serum of affected slightly.

Fig. 10 shows the analysis of the complex formation of variant 1041-D11 heterodimers of ubiquitin fragments of fibronectin due to the CO-GHWR.

Fig. 10A shows the formation of a complex of 1041-D11 with ED-B. Three chromatograms GHWR superimposed on each other: the blue peak with a retention time of 21,651 min is pure 1041-D11; black peak with a retention time of 26,289 min represents the fragment of fibronectin 67 8 9; a mixture of 1041-D11 and 67 8 9 gives red peak with a retention time of 21,407 min after CO-GHWR. The shift of the peak of 1041-D11 to shorter retention time, as well as ischeznovenie� peak 67 V89 indicates the formation of a complex of 1041-D11 soluble and ED-B.

Fig. 10 shows an overlay of three chromatograms CO-GHUR 1041-D11 (blue, 21,944 min), the fragment of fibronectin 6789 without ED-B (black, 26,289 min) and a mixture of 1041-D11 and 6789 (red line with peaks in 21,929 26,289 mines and min). Almost not observed shift of the peak of 1041-D11. This fact together with the lack of disappearance of the peak 6789 indicates a slight binding of the free fragment of fibronectin 6789 ED-B.

Fig. 11 shows a consensus position and amino acid substitution variants binding to ED-B. 16 Shows representative sequences heterodimers that have been found to have a surprisingly strong binding affinity to ED-V. the provisions of the Consensus amino acids in the first monomer region, which determines the binding are 2, 4, 6, 62, 63, 64, 65, 66, and consensus substitutions of amino acids are Q2T, F4W, K6H, Q62N, K63F, E64K, S65L, and T66S.

Fig. 12 shows the sequence alignment of the six heterodimeric proteins through the ubiquitin binding MIA2. The second monomer of ubiquitin begins with methionine at position 89 (1111-B4,1111-C9) or at position 80 (1111-E10, 1111-F6 P-H12,1111-H2).

Fig. 13 shows the alignment of the field that defines the binding OSD and OSD and linkers linking MIA2 heterodimeric proteins on the basis of ubiquitin to Fig. 12. Also shown additional substitutions of amino acids in the sequence of ubiquitin.

Fig. 14 shows W�hanging on the concentration of the ELISA binding of variant 1111-E 10 of Fig. 12 biotinylating MIA-2 (biot. MIA2), Kd=2.6 µmol (shaded circles); control of human serum albumin (HSA) (unfilled circles).

Fig. 15A. Modifications were made in amino acid residues in a series of molecules of the first and second Monomeric ubiquitin units, and sequence alignment were performed to assess the strongest binding sites. Part a shows the information on the sequence for the first, and Part b for the second Monomeric link of the modified ubiquitin.

Fig. 15V. Modifications at positions 2, 4, 6, 62-66, 68 of the first monomer of ubiquitin and in regulations 6, 8, 62-66 in the second monomer. The linker between the two ubiquitinate monomers: SGGGGSGGGGIG.:

Fig. 15S.Shown concentration-dependent ELISA of the binding of the variant SPWF-15_6-A12 heterodimers of ubiquitin from human TNF-alpha. Binding protein SPWF-15_6-A12 shows the binding with very high affinity to TNF-alpha (Kd=12 nm=1,2×10-8M). This figure shows the binding with a high affinity for human TNF-alpha (shaded circles); control BSA (unfilled circles).

Fig. 15D. Sequence binding protein SPWF-15_16-D4_Th heterodimers of ubiquitin with specificity to TNF-alpha. Modifications at positions 2, 4, 6, 62-66 of the first monomer of ubiquitin and in regulations 6, 8, 62-66 in the second monomer. The linker between the two ubiquitin monomers�: SGGGGSGGGGIG.

Fig. 15TH. Shown concentration-dependent ELISA of the binding of the variant SPWF-15_16-D4_Th heterodimers of ubiquitin from human TN Falpha. Binding protein SPWF-15_6-A12 shows a very high affinity to TNF-alpha (Kd=1,7 nm=1,7×10-9M). This figure shows the binding of human TNF-alpha (shaded circles); control: bovine serum albumin (BSA) (unfilled circles).

Fig. 16 shows the binding of NGF by heterodimers on the basis of the modified ubiquitin.

Fig. 16A. Sequence binding protein SPWF9-1B7-th with specificity for NGF. Modifications at positions 2, 4, 6, 62-66 and in position 51 of the first monomer of ubiquitin and in regulations 6, 8, 62-66 in the second monomer. The linker between the two monomers of ubiquitin: SGGGGSGGGGIG.

Fig. 16B. Concentration-independent ELISA determines binding with high affinity Kd of 0.9 μm=9×10-7M) with NGF. The figure shows the binding of recombinant-tion of human NGF (rhNGF; shaded circles); control: BSA (unfilled circles).

Fig. 16C. Sequence binding protein SPWF9-6A2-th heterodimers of ubiquitin with specificity for NGF. Modifications at positions 2, 4, 6, 62-66 of the first monomer of ubiquitin and in regulations 6, 8, 62, 64-66 in the second monomer. The linker between the two monomers of ubiquitin: SGGGGSGGGGIG.

Fig. 16D. Concentration-independent ELISA determines binding with high affinity Kd of 180 nm=1,8×10- M) with NGF. The figure shows the binding of recombinant nye human NGF (rhNGF; shaded circles); control: BSA (unfilled circles).

Fig. 17 Heterodimeric proteins that bind IgG.

Fig. 17A. Sequence binding protein SPVF4-16B2-ts heterodimers of ubiquitin with specificity for IgG. Modifications at positions 6, 62, 63, 65, 66 of the first monomer of ubiquitin and in regulations 6, 62-66 in the second monomer. The linker between the two monomers of ubiquitin: SGGGGSGGGGIG. Fig. 17 V. concentration-Dependent ELISA determines the binding affinity with Kd of 3.8 μm with IgG. The figure shows the binding of IgG (filled black circles); control: BSA-1, BSA-2 and Enbrel (red, green and blue circles without fitted line). Enbrel is Fe part of human IgG1. Weak binding to Enbrel indicates the binding SPVF4-16B2-ts with part FabIgG.

Fig.17C. Sequence binding protein SPVF4-9C6-ts heterodimers of ubiquitin with specificity for IgG. Modifications in regulations 6, 8, 62-66 of the first monomer of ubiquitin and in regulations 6, 8,62-66 in the second monomer. The linker between the two monomers of ubiquitin: SGGGGSGGGGIG. Fig. 17D. Concentration-independent ELISA determines the binding affinity with Kd of 4.1 μm with IgG. The figure shows the binding of IgG (filled black circles); control: BSA (unfilled circles) and Etanercept (brand name Enbrel) (red circles without fitted line). Enbrel carries the Fc portion of human IgG1. Weak binding to Enbrel indicates the binding SPVF4-9C6-ts with part FabIgG.

EXAMPLES

The following Examples are provided to further illustrate the invention. The invention, in particular, demonstrated in relation to the modification of ubiquitin as an example. The invention, however, it is not limited to, the following Examples will just show the feasibility of the invention in practice on the basis of the above description. For full disclosure of the invention reference is also made to the publications mentioned in this application and in the application, are all incorporated in full into this application by reference.

Example 1. Identification heterodimeric proteins, binding to ED-B, on the basis of modified ubiquitin proteins

Designing and planning of the library

Unless otherwise noted, use a well-known recombinant genetic methods, which are described, for example, in the publication of Sambrook and others (Sambrook et al).

Nonspecific library heterodimers of human ubiquitin with high complexity was prepared by concerted mutagenesis of in total 15 selected provisions of amino acids.A modified amino acid which have been replaced by NNK triplets, comprised of at least 3 amino acids selected from Polo�eny 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 in the proximal (first) monomer of ubiquitin and at least 3 amino acids selected from the provisions 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 in the distal (second) monomer of ubiquitin. Both monomers of ubiquitin were genetically related (head to tail) the linker glycine/serine at least with the sequence GIG or a linker glycine/serine at least with sequence SGGGG, for example GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG (SEQ ID NO:32) or SGGGGSGGGG, but any other possible the linker.

Selection of phage display TAT

Library heterodimers of ubiquitin was enriched on the use of the target, for example, TAT phage display as the system of choice. Can be used other ways, known in this field. The target can be immobilized on nonspecific binding surfaces of the protein or via biotinylated residues that were covalently linked to protein. The preferred immobilization via Biotin on the pellet streptavidin or strips neutravidin. Phage binding to the target, either in solution or immobilized target; for example, biotinylated and immobilized target with phage were incubated by washing phages associated with the matrix, and by elution of phages associated with the matrix. In each cycle after incubation of the target pellets otdelalis solution magnetically and washed several times. In the first cycle of selection biotinylation target was immobilized on strips neutravidin, whereas in cycles 2-4 were performed elections in solution, followed by immobilization of complexes of the target-phage coated with streptavidin on the granules Dynabeads® (Invitrogen). After washing in the first two cycles of selection of phages binding to the target of the modified ubiquitin was released by elution with an acidic solution. In cycles 3 and 4 elution of phages was performed by competitive elution with excess target. - Eluted phages were reamplification. For directions to the specificity of the binder during the selection, you can include protein, similar to the target.

Alternative choice TAT phage display: a selection of ribosomal display

Library of ubiquitin was enriched on the use of the target, for example, ribosomal display as the system of choice (sand, etc. (Zahnd et al.), 2007), Ohashi and others (Ohashi et al.), 2007). Can be used other ways, known in this field. The target is biotinylated according to standard methods and immobilized on coated with streptavidin pellets Dynabeads®. (Invitrogen company). The ternary complexes, including the ribosome, mRNA and emerging ubiquitinates polypeptide, were assembled using the kit PURExpress™ In Vitro protein synthesis (NEB). Were in�made two primary cycle choice in which were incubated ternary complexes, after which howled executed two similar cycle of selection. In each cycle after incubation of the target pellets were separated from the solution by magnetic separation and washed with buffer ribosomal display with increasing thoroughness. After washing in the first two cycles of selection granules are again separated from the solution magnetically, and mRNA molecules of the modified ubiquitin that communicates with the target, was released from ribosomes by adding. 50 mm EDTA. In cycles 3 and 4 select elution of mRNA was carried out by competitive elution with excess target (Lipovsek and Planton (Lipovsek and Pluckthuh), 2004). After each cycle performed the purification of RNA and synthesis of cDNA using the kit RNeasy MinElute Cleanup Kit (Qiagen company, Germany), set "Turbo DNA-free Kit (Applied Biosystems, USA) and reverse transcriptase inhibitors of TRANS-Cryptor (Roche company, Germany).

Cloning of enriched pools

After the fourth cycle of selection of the synthesized cDNA was amplified by PCR according to the method, cut with suitable rest room riccioni nucleases and Legerova in expressing vector pet-20b(+) (Merck, Germany) using a compatible sticky ends.

The analysis of coincidences cloned colonies

After transformation the cells NovaBlue(DE3) (Merck, Germany) resistant to ampicillin kleinova�nye colonies were grown for 6 h at 37°C in 200 µl of SOBAG medium (SOB medium, containing 100 µg/ml ampicillin and 20 g/l glucose), expression of the modified ubiquitin that binds to ED-B was achieved by culturing for 16 h at 37°C in 96-well deep plates (the company Genetix, UK) using 500 ál autoinduction medium ZYM-5052 (Studier (Studier), 2005). Cells were collected and then lisarova. After centrifugation, the resulting supernatants were subjected to screening by ELISA, using tablets Nunc MediSorp (Thermo Fisher Scientific, USA) coated with 4 µg/ml ED-B and ubiquitin-specific Fab fragment, conjugio-bathrooms with horseradish peroxidase. As detecting reagent used TMB-Plus (Biotrend, Germany), and the yellow color was obtained, using 50 µl/well of 0.2 M H2S04 solution, measured in the device for reading tablets at 450 nm against 620 nm.

Usually performed several cycles of selection display against targets. In the last two cycles of selection of binding molecules has suirable with excess free of the target.

For example, have been identified heterodimerize modified ubiquitin-ries binding proteins against the target ED-B, such as N (SEQ ID NO:6), E (SEQ ID NO:7), 22D1 (SEQ ID NO:8), 1041-D11 in Fig. 5 (SEQ ID NO:33), 1045-D10 (SEQ ID NO:34). For example, have been identified heterodimerize modified ubiquitinate binding proteins against different targets, for example, against a protein 1111-E10 Fig. 12 (SEQ ID O:53), linking target MIA-2, against proteins SPWF-15_6-A12 of Fig. 15 (SEQ ID NO:57) and SPWF-15J6-D4 of Fig. 15D(SEQ ID NO:90), which links the target TNF-alpha, against proteins SPWF9-1B7-th Fig. 16A (SEQ ID NO:91) and SPWF9-6A2-th Fig. 16C (SEQ ID NO:92) connecting the target and against NGF protein SPVF4-16B2-ts Fig. 17A (SEQ ID NO:93) and SPVF4-9C6-ts Fig. 17C (SEQ ID NO:94), which links the target IgG.

Sequence alignment of the monomer of wild-type ubiquitin (UBI-dt monomer) (dimer of wild-type ubiquitin (UBI-dt dimer) and ubiquitinated protein (Ub2-TsX Fig. 5 with the substitution at position 45 of each monomer and with two substitutions in the C-end) with heterodimer variant 1041-D11 modified ubiquitin is shown in Fig. 5. In Ub2-TsX substitution in the C-graduation (GG to AA) monomer enhance the stability in serum, as deubiquitinase disintegrate in GG of ubiquitin, but not for AA. The secondary structure of ubiquitin wild type compared to ubiquitin with these substitutions in the C-end is almost identical.

Modified ubiquitin with an activity of binding to ED-B, as specified 1041-D11 (shown in Fig. X; SEQ ID NO:36) or 1045-D10 are identified by the following substitutions of amino acids compared to the wild type: in the first module: K6W, L8W, K63R, EC, S65F, TR; in the second module: CBT, L8Q, Q62W, K. 63S, E64N, S65W, TE; Q2R (in the variant 1041-D11, but not in variant 1045-D10). Suitable preferred linkers for this protein composite are linkers having at least the sequence GIG or having at least sequence SGGGG, or any other linker, for example, GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG or SGGGGSGGGG. However, there are many known linkers that can be used. Other substances that bind EDB, with their consensus sequence in the first Monomeric region that defines the binding shown in Fig. 11.

Modified ubiquitin with excellent binding activity MIA-2 shown in Fig. 12-14.

Modified ubiquitin with an activity of binding of NGF is shown in Fig. 16.

Modified ubiquitin with an activity of binding TNF-alpha shown in Fig. 15.

Modified ubiquitin with excellent binding activity of IgG is shown in Fig. 17.

Example 2: Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin with a human target

Example 2A. Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin by ELISA, concentration-independent.

Linking options on the basis of ubiquitin to the target person were analyzed by ELISA, which depends on concentration. Increasing amounts of purified protein were applied to the plates NUNC-medisorp covered by the target person, BSA or serum human albumin (SAC) and other possible controls, such as cellular fibronectin (cFN), if the target has used ED-B. the Antiga�Noe coated with 50 μl (10 μg/ml) of protein solution per well was performed at 4°With during the night. After washing tablets of phosphate-saline buffer (FSB), 0,1% Tween-20, pH 7,4 (FSBT) the wells were blocked using a blocking solution (PBS pH 7.4; 3% BSA; 0.5% of Tween-20) at room temperature for 2 h. the Wells are again three times washed FSBT and then three times with PBS. Coated wells were incubated with different concentrations of protein binding to a target, at room temperature for 1 h. After washing the wells FSBT struck fragment conjugates anti-Ubi fab (AbyD) POD in a suitable dilution in FSBT. The plate was washed three times with buffer FSBT. To each well was added 50 µl of TMB substrate solution (KEM-EN-TEC) and incubated for 15 min the Reaction was stopped by adding 0.2 M H2SO4. Tablets ELISA was read using a reader ELISA TECAN Sunrise. Photometric measurements of the spectral absorptivity performed at 450 nm using 620 nm as reference wavelength. Fig. 6 shows the binding with a high affinity variant 1041-D11 to ED-B (Kd=6,9 nm). This is confirmed in relation to other target molecules MIA-2, TNF-alpha, NGF IgG and the results shown in Fig. 14, 15, 16 and 17, respectively. Thus, only a few modifications (up to 8 substitutions in each monomer) in wild-type ubiquitin give affinity data targets in the low micromolar range.

Example 2B. Analysis of binding options, binding ED-B,on the basis of the modified ubiquitin by competitive ELISA, concentration-independent.

Here we describe the analysis of binding to the target ED-B, but without further experiments, it can be used for any other target. Was made competitive, concentration-dependent ELISA for the analysis of binding options 1041-30 D11 of ubiquitin with immobilized ED-B containing a fragment of fibronectin (67 8 9), in the presence of increasing amounts of free target. The ELISA conditions were as described for Example 2A, except that the protein 1041-D11 was pre-incubated with ED-B (67 To 89) (0 μm - 10 μm) or with negative control 6789 (0 μm - 10 μm) for 1 h and then the mixture with the target 67 V89 placed on the tablet Medisorp; after that this variant was detected corresponding antibody (anti-Ubiquitin-Fab-horseradish peroxidase; dilution of 1:6500).

Fig. 7 shows that the variant 1041-D11 has a very high-affinity binding to ED-B (IC50=140 nm). The result, shown in Fig. 6 confirmed; only a few modifications (up to 8 substitutions in each monomer) in wild-type ubiquitin lead to very high-affinity binding to ED-B.

Example 2C. Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin by ELISA, which depends on concentration, with simultaneous analysis of the stability of the binding activity in serum.

ELISA was performed using SP�events, well known in this area as described above (Example 2A and 2B). ED-B (here named 67 V89) inflicted on microtic-quotation tablets, this variant was associated with ED-B and were detected by the antibody specific to ubiquitin (anti-Ubi-Fab-horseradish peroxidase). Option in this analysis were treated in different ways: variant were incubated in the serum of mice for 1 h at 37°C (see Fig. 9, blue circles); variant were incubated in serum of rats within for 1 h at 37°C (Fig. 13X red circles), or were incubated in PBS for 1 h at 37°C (Fig. 9 black circles). Fig. 13 shows that all the KD of the variant 1041-D11 ranged from 10.3 nm (in PBS) to 20,74 nm (in mice serum).

Example 2D. Analysis of binding options, binding ED-B, on the basis of the modified ubiquitin by Biacore analysis.

Were analyzed variants of different concentrations (e.g. 0 - 200 variant nm, preferably 1041-D11) for binding to ED-B containing a fragment of the fiber-nectin (named 67 V89) immobilized on the cm 5 chip (Biacore) using methods known to experts in this field. The obtained data were processed by the software BIAevaluation and 1:1-Langmuir-fit. The molecular weight of the variant 1041-D11 was 1.0 nm, as shown in Fig. 8. Kinetic binding constants amounted to kon=7,6*105M-1with-1; koff =7,7*10-4c-1. kdcomposite protein 1041-D11 - TNF-alpha amounted to 1.13 nm. Kinetic binding constants amounted to kon=4,5*105M-1with-1; koff=5,0*10-4c-1.

Example 2E. Analysis of the formation of the complex variants that bind ED-B, on the basis of the modified ubiquitin by CO-GHWR.

For analysis of the complex formation used column Tricorn Superdex 75 5/150 GL (GE-Healthcare) (volume =3 ml), number of input protein was 50 µl. Other conditions: buffer: 1×PBS, pH 7,3, flow rate: 0.3 ml/min, cycle: 45 min (injection of the sample after 15 min). Condition: 0,72 nmole protein 1041-D11+0,72 nmole ED-B (here called W or negative control 6789) were incubated for 1 h at room temperature; then put on the column for analysis of complex formation. Fig. 10 only variant is shown in black, only the target ED-B is shown in blue, binding variants, forming a complex with ED-B, shown in pink. Fig. 10A shows fibronectin containing the ED-B with that option; Fig. 10 shows a variant without fibronectin ED-B(6789). This Figure shows that the variant 1041-D11 forms a complex with ED-B(V), but does not form a complex with fibronectin (6789), confirming specificity.

Example 3: Heterodimer binding proteins on the basis of ubiquitin with improved binding to TNF-alpha

Heterodimerize svyazyvayus� proteins on the basis of ubiquitin, specific for TNF-alpha, were selected according to the method of the present invention, i.e., was established phage library, which included a set of modified heterodimeric ubiquitin binding proteins that were subjected to screening for their potential binding to TNF-alpha. Performed the following modifications:

in the first monomer, one or more amino acids in positions 2, 4, 6, 62-66, optionally additionally one or more of the provisions of 68, 70, 72-74, optionally in the subsidiary arrangements;

in the second monomer: modification in one or more of the amino acids in positions 6, 8, 62-66.

As the linker in most cases used SGGGGSGGGGIG, except 1144-D11 (SEQ ID NO:79) and 1144-E9 (SEQ ID NO:80). The linker is not used for 1144-D11 and 1144-E9 between the first and second monomer of ubiquitin. Regulations 75 and 76 or AA, or GG. The linker is shown in part A in Fig. 15. Binding affinity is shown in Fig. 15V-E.

Example 4: Create heterodimeric binding proteins on the basis of ubiquitin with improved binding MIA2

MIA2 is a diagnostic and therapeutic marker, inter alia in the context of cirrhosis, fibrosis and liver cancer. Detailed information on this marker can be found in document US 2004076965.

Protein is a target for modified ubiquitin binding proteins of the present izobreteny� is stable the main area MIA-2 of 101 amino acids, referred to here as SPR30-3. SPR30-3 is a structured part of MIA-2. She is homologous to MIA (CD-RAP), OTOR, TANGO, excluding the signal peptide. Its molecular weight is 11569,198 Yes.

Basic amino acid region MIA-2 is as follows (SEQ ID NO:95):

MLESTKLLADLKKCGDLECEALINRVSAMRDYRGPDCRYLNFTKGEEISVYVKLAGE REDLWAGSKGKEFGYFPRDAVQIEEVFISEEIQMSTKESDFLCL

Heterodimer binding proteins through the ubiquitin-specific MIA2, were selected according to the method of the present invention, i.e., was established phage library, which included a set of modified heterodimeric ubiquitin binding proteins that were subjected to screening for their potential binding MIA2. The results are as follows.

Fig. 13 shows the alignment heterodimeric proteins binding MIA2, on the basis of ubiquitin.

Variant 1111-E10 shows an affinity in the micromolar range biotinidase-bath of the target and the formation of the complex in exclusion chromatography. The strongest binding substance labeled 1111-E10 with replacements of amino acids at positions 6, 8, 62, 63, 64, 65, 66 in the first Monomeric unit of ubiquitin (OSD) and different substitutions at positions 6, 8, 62, 63, 64, 65, 66 in the second Monomeric unit of ubiquitin (OSD).

The first monomer unit of ubiquitin (OSD) shows the same substitution as in 1111-H2 and 1111-H12. Options 111 1-H2 and 1111-H12 may therefore be considered a combination of the OSD and OSD, which differ by only one substituted amino acid.

Were evaluated the following other strong binding molecules: 1111-C9, 1111-B4 and 1111-F6. These binding molecules or were insoluble, or showed no binding to SPR30-3 MIA2 ELISA and chromatography with a step displacement. Options 1111-E10 and 1111 - C9, respectively, and 1111-B4 were enriched (additional substitution TO in 1111-B4 happened several times). Option 111 1-F6 was not enriched, but seemed a good candidate because of its high signal in ELISA; however, it was insoluble.

FIG.14 shows concentration-dependent ELISA of the variant 1111-E10, communicating with biotinylating MIA-2 (Biot.MIA2), Kd=2.6 mm (filled circles); control SAC (unfilled circles). This option 1111-E10 proved to be the best molecule that communicates with the MIA2. The sequence is as follows:

MQIFVETFTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDY

NIGWHPELHLVLRLRGGGIGMQIFVRTETGKTITLEVEPSDTIENVKAKIQDKEGIPPD

QQRLIWAGKQLEDGRTLSDYNILMGYVLHLVLRLRAA

(SEQ ID NO:53)

Used linkers shown in the attached sequence listing as:

1111-B4_21231sggggsggggigSEQ ID NO:96
1111-SsggggsggggigSEQ ID NO:96
1111-E gig
1111-F6_21331gig

1111-H12_21391 eig
1111-N gig

PUBLICATIONS

1. Birchler, M., F. Viti, L. Zardi, B. Spiess, and D. Neri. 1999. Selective targeting and photocoagulation of ocular angiogenesis also been other ideas where by a phage-derived human antibody fragment. Nat Biotechnol 17:984-8.

2. Brenmoehl, J., M. Lang, M. Hausmann, S. N. Leeb, W. Falk, J. Scholmerich, M. Goke, and G. Rogler. 2007. Evidence for a differential expression offibronectin splice forms ED-A and ED-B in Crohn's disease (CD) mucosa. Int J Colorectal Dis 22:611-23.

3. Dubin, D., J. H. Peters, L. F. Brown, B. Logan, K. C. Kent, B. Berse, S. Berven, B. Cercek, B. G. Sharifi, R. E. Pratt, and et al. 1995. Balloon catheterization induced arterial expression of embryonic fibronectins. Arterioscler Thromb Vase Biol 15:1958-67.

4. Goodsell, D. S. 2001. FUNDAMENTALS OF CANCER MEDICINE: The Molecular Perspective: Antibodies. The Oncologist 6:547-548.

5. Kaczmarek, J., P. Castellani, G. Nicolo, B. Spina, G. Allemanni, and L. Zardi. 1994. Distribution ofoncofetal fibronectin isoforms in normal, hyperplastic and neoplastic human breast tissues. Int J Cancer 59:11-6.

6. Menrad, A., and H. D. Menssen. 2005. ED-B fibronectin as a target for antibody-based cancer treatments. Expert Opin Ther Targets 9:491-500.

7. Pujuguet, P., A. Hammann, M. Moutet, J. L. Samuel, F. Martin, and M. Martin. 1996. Expression offibronectin ED-A+ and ED-B+ isoforms by human and experimental colorectal cancer. Contribution of cancer cells and tumor-associated myofibroblasts. Am J Pathol 148:579-92.

8. Trachsel, E., M. Kaspar, F. Bootz, M. Detmar, and D. Neri. 2007. A human mAb specific to oncofetal fibronectin selctively targets chronic skin inflammation in vivo. J Invest Dermatol 127:881-6.

9. Van Vliet, A., H. J. Baelde, J. L. VIeming, E.de Heer, and J. A. Bruijn. 2001. Distribution offibronectin isoforms in human renal disease. J Pathol 193:256-62.

10. Lipovsek, D., and Pluckthun, A. (2004). In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51-67.

11. Ohashi, H., Shimizu, Y., Ying, B. W., and Ueda, T. (2007). Efficient protein selection based on ribosome display system with purified components. Biochem Biophys. Res. Commun. 352,270-276.

12. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41, 207-234.

13. Zahnd, C., Amstutz, P., and Pliickthun, A. (2007). Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Cnoco6s 4, 269-279.

14. Paschke, M. and W. Hohne (2005). Gene 350(1): 79-88

15. Briiser 2007 Appl Microbiol Biotechnol 76(1): 35-45

1. Method of identification heterodimeric modified ubiquitin with the ability to bind to ligand-antigen, comprising the following stages:
(a) providing aggregate heterodimeric modified by ubiquitin originating from Monomeric modified ubiquitin proteins comprising two monomer of ubiquitin linked scheme head to tail, in which each of the monomers contains 5-8 overrides the provisions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO:1, and referred to a modified Monomeric protein has amino acid sequence identity of at least 80% or at least 90% or at least 95% with unmodified ubiquitinated protein;
(b) providing a potential ligand for the aggregate of differently modified proteins;
c) the contact mentioned together differently modified proteins with said ligand of display method;
(d) identification heterodimers of the modified protein by a screening process, and mentioned modified heterodimeric protein is bound with said ligand with a specific binding affinity
Kd in the range of 10-7-10-12M and exhibits a monovalent binding activity with respect to the above ligand; and n� choice,
e) isolation mentioned heterodimeric modified ubiquitin with said affinity binding,
moreover, the aforementioned monomers are differently modified by substitutions of surface amino acids in the open positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO:1.

2. A method according to claim 1, characterized in that said modified Monomeric protein includes from 1 to 10 insertions of amino acids in the monomer molecule and/or from 1 to 7 deletions of amino acids in the monomer molecule.

3. A method according to any one of the preceding paragraphs, characterized in that said modified Monomeric ubiquitinate protein is produced by genetic engineering of the DNA responsible for the receipt of ubiquitin, and the expression of the mentioned protein in prokaryotic or eukaryotic organisms or in vitro.

4. A method according to claim 3, characterized in that said dimeric protein produced by phage display, ribosomal display, TAT phage display, yeast display, bacterial display, display the cell surface or by way of the display of mRNA.

5. A method according to claim 1, characterized in that from 1 to 7 additional amino acid replaces at least one Monomeric ubiquitin proteins, which are selected from one or more amino acids in positions 8, 36, 44, 62, 63,64, 70, 71, 72 and 73 of SEQ ID NO:1.

6. A method according to claim 1, characterized in that the mentioned�utuu a set heterodimeric protein component of ubiquitin produced by genetic fusion of two DNA libraries each of which is responsible for receiving differently modified Monomeric proteins, translation of this DNA in heterodimerize integral proteins mentioned display protein display and screening of proteins modified by the presence of heterodimeric ubiquitin proteins, including Monomeric ubiquitinate proteins, connected according to the scheme head to tail, and characterized in that said modified heterodimerize ubiquitinate proteins bind with said ligand with a specific binding affinity of Kd in the range of 10-7-10-12M and exhibit monovalent binding activity with respect to the above ligand, or characterized in that said set of heteropolymeric protein component of ubiquitin obtainable by chemical synthesis of proteins.

7. DNA library for use in the method according to claim 1, responsible for receiving the aggregate heterodimeric ubiquitin protein component derived from the monomer of ubiquitin, each dimeric protein comprises two modified monomer of ubiquitin, interconnected according to the scheme head to tail, characterized in that said monomers are differently modified by substitutions of surface amino acids in the open 5-8 amino acids located in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO:1, moreover, by mentioning�th modified Monomeric protein has amino acid sequence identity of at least 80%, or at least 90% or at least 95% with the unmodified protein.

8. A library of proteins for use in the method according to claim 1, obtained by expression of DNA-library according to claim 7.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented a composition and a method for producing it. The characterised composition contains: an effective amount of a viral antigen, which represents a live attenuated rotavirus pre-processed in 0.1% human serum albumin, and a pharmaceutically acceptable buffer. A method for producing a composition involves growing Vero cell culture pre-cultured in the presence of 5% foetal calf serum and 0.1% human serum albumin, infecting the above Vero cell culture with the live attenuated rotavirus, propagating the virus in the cell culture and adding a pharmaceutically acceptable buffer to the above virus.

EFFECT: presented inventions can be used to prevent rotavirus infection and/or rotavirus gastroenteritis.

11 cl, 17 dwg, 4 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.

EFFECT: characterised inventions can be used to prevent infection, caused by porcine Torque teNo virus.

23 cl, 53 dwg, 5 tbl, 24 ex

FIELD: biotechnology.

SUBSTANCE: characterised strain was isolated from diseased pigs and produced by serial passages on sensitive hetero- and homologous cell cultures and deposited in the collection of the FSBI "Federal Animal healthcare centre" under the registration number of FMD virus strain A No.2187/Kuti/2013 (production). The presented strain is reproduced in monolayer cell culture of porcine kidney (PK), passaged cell cultures of kidney of Siberian mountain ibex (SMIK-30), VPK-21 and IB-RS-2. During 18÷24 hours of incubation the virus yield in the said cell cultures reaches the values of 6.0÷7.0 lg TCD50/cm3. At high multiplicity of infection (1÷10 TCD/cell) causes TCID50 after 5 hours, while maintaining the original characteristics when passaging in cell cultures for 5 passages.

EFFECT: invention can be used to monitor the antigenic and immunogenic activity and for producing biological products for diagnostics and specific prophylaxis of FMD of type A.

6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology. The subject of the invention is a new strain of the Sendai virus Sen293nsk1 adapted to effective replication in the human cell culture HEK293. The produced strain of the Sendai virus possesses lower virulence for laboratory mice and higher ability to destroy human tumour cells. The strain is supposed to be used experimentally as a therapeutic oncolytic preparation for treating malignant diseases. The invention can be used in treating oncologic diseases.

EFFECT: invention enables providing higher clinical effectiveness in oncologic diseases by using the murine Sendai virus non-pathogenic for humans, possessing an increased oncolytic activity and intensifying anti-tumour immunity.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.

EFFECT: obtaining pharmaceutical compositions, which include poxviruses.

11 cl, 3 dwg

FIELD: biotechnology.

SUBSTANCE: method of detection and differentiation of a genome of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates comprises: extraction of the DNA from the biological material, posing a multiplex polymerase chain reaction using synthesised primers complementary to regions of genes M130R and M151R of the myxoma virus of rabbit, and having the following nucleotide composition: MF 5'TGG-AGC-TTT-TCA-AGC-ATT 3', MR 5'ATA-TCT-CGG-CTC-TAG-GGC-GAG 3', MZ 5' [FAM]-AG-CGT-CGG-ACG-TCT-TCG-TT-[RTQ1] 3', VF 5'AGC-CCT-ATA-AAC-CCG-TAG-ACG-AAC 3', VR 5'CAA-GCT-TTT-TTT-TAT-CCT-CGT-CCG 3', VZ 5' [R6G]-TCG-ACG-GTT-TCG-TCC-GCC-TTC-TTG-[BHQ2] 3', DNA amplification of the virus and evaluation of the reaction. The present inventions may be used in veterinary virology for the detection and differentiation of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates.

EFFECT: increase in accuracy.

2 cl, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.

EFFECT: preparing the new avian astrovirus.

11 cl, 5 dwg, 4 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing influenza A or B viruses in a cell culture, and a composition of the cell culture for preparing influenza A or B viruses.

EFFECT: invention provides the serum-free culture medium, avoids the need for the stage of cell culture medium replacement, prepares the influenza viruses with the high live virus recovery and can be used for the active immunisation of individuals and for producing the antibodies for various applications, including passive immunisation and diagnostic immunoassays.

22 cl, 24 dwg, 47 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for producing viral-like influenza virus particles (IVP) in a plant or a part thereof. The method involves the expression of a new influenza virus protein HA in plants and purification thereof. The invention also aims at IVPs containing the influenza virus protein HA and herbal lipids. The invention also refers to nucleic acids coding an improved influenza virus HA, as well as to vectors. The IVPs can be used in developing the influenza vaccines or for the treatment of existing vaccines.

EFFECT: presented group of inventions can be used in medicine.

18 cl, 44 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with an influenza virus strain. The vaccine strain B/60/Massachusetts/2012/10 is a reassortant, obtained by crossing the "wild" virus B/Massachusetts/2/2012 with the cold-adapted temperature-sensitive virus B/USSR/60/69 -attenuation donor. The strain B/60/Massachusetts/2012/10 is deposited in the State Collection of Viruses FSBI D.I.Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health under No 2740, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (HA) and neuraminidase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza morbidity among adults and children.

4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. There are presented versions of antibodies neutralising a subtype group 1 and subtype group 2 influenza A virus infection. The antibody is characterised by: either a set of 3 CDR of a light and 3 CDR of a heavy chain, or the presence of variable regions of the light and heavy chains. There are disclosed: a nucleic acid molecule coding the antibody; a cell expressing the antibody; as well as a method for the attenuation of the influenza A virus infection or reducing a risk thereof with the use of the antibody in a therapeutically or preventatively effective amount.

EFFECT: using the invention provides the antibodies neutralising the influenza A virus, which can find application in medicine in treating subtypes H1, H2, H3, H5, H7, H9 influenza.

18 cl, 6 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.

EFFECT: characterised inventions can be used to prevent infection, caused by porcine Torque teNo virus.

23 cl, 53 dwg, 5 tbl, 24 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.

EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.

2 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and gene engineering. A method for selecting at least one transfected eukaryotic host cell expressing a target product, the eukaryotic host cells comprise at least an introduced polynucleotide encoding the target product, an introduced polynucleotide encoding a DHFR enzyme using at least one expression vector, providing a plurality of eukaryotic host cells, whose viability is dependent upon folate uptake, wherein the said host cells comprise at least a foreign polynucleotide encoding the target product, a foreign polynucleotide encoding a DHFR enzyme, culturing the said plurality of the eukaryotic host cells in a selective culture medium comprising folic acid in a concentration of 12.5-50 nM combined with a concentration of MTX of 2.3-500 nM, selecting at least one eukaryotic host cell expressing the target product.

EFFECT: described is a method of the target product and culture medium preparation.

11 cl, 2 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.

EFFECT: present invention can find further application in cancer therapy.

16 cl, 27 ex, 18 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.

EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.

33 cl, 21 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.

EFFECT: Chinese hamster ovary cell line (CHO) produced by transfection by expression structures containing the genetic sequences according to the invention, enables producing at least 50 mg/l of the monoclonal antibody Infliximab.

4 cl, 3 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology and represents anti-nerve growth factor (NGF) antibodies. The present invention also discloses a pharmaceutical composition for relieving pain associated with a disease or a condition, wherein pain progression or persistence is mediated by NGF, containing the above antibodies, as well as a kit for treating a HGF-related disease, such as e.g. osteoarthritis, nucleic acids coding a heavy or light chain of the antibody, an expression vector, a host cell for preparing the above antibodies, a method for expressing the above anti-NGF antibodies, as well as using the above antibodies in managing pain and for preparing a therapeutic agent for managing pain associated with the disease or condition, wherein pain progression or persistence is mediated by NGF.

EFFECT: present invention enables producing the anti-NGF antibodies characterised by high stability in vivo.

16 cl, 7 dwg, 13 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.

EFFECT: invention can find further application in diagnosing and treating cancer.

43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: biotechnology.

SUBSTANCE: synthetic DNA is proposed, encoding human erythropoietin, having the sequence Seq ID No. 1, comprising its expression vector, the method of production of erythropoietin producer strain, and a strain of a Chinese hamster ovary cells - producer of recombinant human erythropoietin, deposited under the number RKKK(P) 761 D.

EFFECT: invention enables to increase the expression level of recombinant human erythropoietin.

5 cl, 1 tbl, 8 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.

EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.

2 dwg, 3 ex

Up!